Hybrid sweet corn plant named AZLAN

ABSTRACT

A novel hybrid sweet corn plant, designated AZLAN is disclosed. The invention relates to the seeds of hybrid sweet corn designated AZLAN, to the plants and plant parts of hybrid sweet corn designated AZLAN, and to methods for producing a sweet corn plant by crossing the hybrid sweet corn AZLAN with itself or another sweet corn plant.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture, to new anddistinctive hybrid sweet corn plants, such as a hybrid plant designatedAZLAN and to methods of making and using such hybrids.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Sweet corn is an important and valuable vegetable crop. Thus, acontinuing goal of plant breeders is to develop stable, high yieldingsweet corn hybrids that are agronomically sound or unique. The reasonsfor this goal are to maximize the amount of ears or kernels produced onthe land used yield as well as to improve the plant, the ears, husks,kernel shape and size, eating and processing qualities and/or the plantagronomic and horticultural qualities. To accomplish this goal, thesweet corn breeder must select and develop sweet corn plants that havethe traits that result in superior parental lines that combine toproduce superior hybrids.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope.

In various embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

According to the invention, in some embodiments there is provided anovel hybrid sweet corn designated AZLAN, also interchangeably referredto as ‘hybrid sweet corn AZLAN’, ‘sweet corn hybrid AZLAN’ or ‘AZLAN’.

This invention thus relates to the seeds of hybrid sweet corn designatedAZLAN, to the plants or parts of hybrid sweet corn designated AZLAN, toplants or parts thereof comprising all the physiological andmorphological characteristics of hybrid sweet corn designated AZLAN orparts thereof, and/or having all the physiological and morphologicalcharacteristics of hybrid sweet corn designated AZLAN, and/or having oneor more of or all the characteristics of hybrid sweet corn designatedAZLAN listed in Table 1 including but not limited to as determined atthe 5% significance level when grown in the same environmentalconditions, and/or having one or more of the physiological andmorphological characteristics of hybrid sweet corn designated AZLANlisted in Table 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditionsand/or having all the physiological and morphological characteristics ofhybrid sweet corn designated AZLAN listed in Table 1 including but notlimited to as determined at the 5% significance level when grown in thesame environmental conditions and/or having one or more of thephysiological and morphological characteristics of hybrid sweet corndesignated AZLAN listed in Table 1 when grown in the same environmentalconditions and/or having all the physiological and morphologicalcharacteristics of hybrid sweet corn designated AZLAN listed in Table 1when grown in the same environmental conditions. The invention alsorelates to variants, mutants and trivial modifications of the seed orplant of hybrid sweet corn designated AZLAN.

Plant parts of the hybrid sweet corn plant designated AZLAN of thepresent invention are also provided, such as, but not limited to, ascion, a rootstock, an ear, a kernel, a leaf, a flower, a peduncle, astalk, a root, a stamen, an anther, a pistil, a pollen or an ovuleobtained from the hybrid plant. The present invention provides ears andkernels of the hybrid sweet corn plant designated AZLAN of the presentinvention. Such ears, kernels and parts thereof could be used as freshproducts for consumption or in processes resulting in processed productssuch as food products comprising one or more harvested parts of thehybrid sweet corn designated AZLAN, such as prepared kernels or partsthereof, canned kernels or parts thereof, freeze-dried or frozen kernelsor parts thereof, diced kernels, juices, prepared kernel cuts, cannedsweet corn, pastes, sauces, powders, purees and the like. All suchproducts are part of the present invention and the like. The harvestedparts or food products can be or can comprise hybrid sweet corn fruitfrom hybrid sweet corn designated AZLAN. The food products might haveundergone one or more processing steps such as, but not limited tocutting, washing, mixing, frizzing, canning, etc. All such products arepart of the present invention. The present invention also provides plantparts or cells of the hybrid sweet corn plant designated AZLAN, whereina plant regenerated from said plants parts or cells has one or more of,or all the phenotypic and morphological characteristics of hybrid sweetcorn designated AZLAN, such as one or more of or all the characteristicsof hybrid sweet corn plant designated AZLAN, listed in Table 1 includingbut not limited to as determined at the 5% significance level when grownin the same environmental conditions. All such parts and cells are partof the present invention.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act of The United States of America, e.g., avariety that is predominantly derived from hybrid sweet corn designatedAZLAN or from a variety that i) is predominantly derived from hybridsweet corn designated AZLAN, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of hybrid sweet corn designated AZLAN; ii) is clearlydistinguishable from hybrid sweet corn designated AZLAN; and iii) exceptfor differences that result from the act of derivation, conforms to theinitial variety in the expression of the essential characteristics thatresult from the genotype or combination of genotypes of the hybrid sweetcorn plant designated AZLAN.

In another aspect, the present invention provides regenerable cells. Insome embodiments, the regenerable cells are for use in tissue culture ofhybrid sweet corn designated AZLAN. In some embodiments, the tissueculture is capable of regenerating plants comprising all thephysiological and morphological characteristics of hybrid sweet corndesignated AZLAN, and/or having all the physiological and morphologicalcharacteristics of hybrid sweet corn designated AZLAN, and/or having oneor more of the physiological and morphological characteristics of hybridsweet corn designated AZLAN, and/or having the characteristics of hybridsweet corn designated AZLAN. In some embodiments, the regenerated plantshave the characteristics of hybrid sweet corn designated AZLAN listed inTable 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditionsand/or have all the physiological and morphological characteristics ofhybrid sweet corn designated AZLAN listed in Table 1 including but notlimited to as determined at the 5% significance level when grown in thesame environmental conditions and/or have one or more of thephysiological and morphological characteristics hybrid sweet corndesignated AZLAN listed in Table 1 including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions and/or have all the physiological andmorphological characteristics of hybrid sweet corn designated AZLANlisted in Table 1 when grown in the same environmental conditions.

In some embodiments, the plant parts and cells used to produce suchtissue cultures will be embryos, meristematic cells, seeds, callus,pollens, leaves, anthers, pistils, stamens, roots, root tips, stems,petioles, kernels, cotyledons, hypocotyls, ovaries, seed coats, kernels,stalks, endosperms, flowers, axillary buds or the like. Protoplastsproduced from such tissue culture are also included in the presentinvention. The sweet corn leaves, shoots, roots and whole plantsregenerated from the tissue culture, as well as the kernels produced bysaid regenerated plants are also part of the invention. In someembodiments, the whole plants regenerated from the tissue culture haveone, more than one, or all the physiological and morphologicalcharacteristics of sweet corn hybrid designated AZLAN listed in Table 1,including but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions.

The invention also discloses methods for vegetatively propagating aplant of the present invention. In the present application, vegetativelypropagating can be interchangeably used with vegetative reproduction. Insome embodiments, the methods comprise collecting parts of a hybridsweet corn designated AZLAN and regenerating a plant from said parts. Insome embodiments, one of the parts can be for example a stem. In someembodiments, the methods can be for example a stem cutting that isrooted into an appropriate medium according to techniques known by theone skilled in the art. Plants and parts thereof, including but notlimited to ears and kernels thereof, produced by such methods are alsoincluded in the present invention. In another aspect, the plants, earsand parts thereof such as kernels thereof produced by such methodscomprise all the physiological and morphological characteristics ofhybrid sweet corn designated AZLAN, and/or have all the physiologicaland morphological characteristics of hybrid sweet corn designated AZLANand/or have the physiological and morphological characteristics ofhybrid sweet corn designated AZLAN and/or have one or more of thecharacteristics of hybrid sweet corn designated AZLAN. In someembodiments, plants, parts or kernels thereof produced by such methodsconsist of one, more than one, or all the physiological andmorphological characteristics of sweet corn hybrid designated AZLANlisted in Table 1, including but not limited to as determined at the 5%significance level when grown in the same environmental conditions.

Further included in the invention are methods for producing ears andkernels and/or seeds from the hybrid sweet corn designated AZLAN. Insome embodiments, the methods comprise growing a hybrid sweet corndesignated AZLAN to produce sweet corn kernels and/or seeds. In someembodiments, the methods further comprise harvesting the hybrid sweetcorn ears. Such ears and their kernels and/or seeds. Such kernels and/orseeds are parts of the present invention. In some embodiments, such earand kernels and/or seeds have all the physiological and morphologicalcharacteristics of ear, kernels and/or seeds of hybrid sweet corndesignated AZLAN (e.g. those listed in Table 1) when grown in the sameenvironmental conditions and/or have one or more of the physiologicaland morphological characteristics of the kernels and/or seeds of thehybrid sweet corn designated AZLAN (e.g. those listed in Table 1) whengrown in the same environmental conditions and/or have thecharacteristics of the kernels and/or seeds of the hybrid sweet corndesignated AZLAN (e.g. those listed in Table 1) when grown in the sameenvironmental conditions.

Also included in this invention are methods for producing a sweet cornplant. In some embodiments, the sweet corn plant is produced by crossingthe hybrid sweet corn designated AZLAN with itself or other sweet cornplant. In some embodiments, the other plant can be a hybrid sweet cornother than the hybrid sweet corn designated AZLAN. In other embodiments,the other plant can be a sweet corn inbred line. When crossed with aninbred line, in some embodiments, a “three-way cross” is produced. Whencrossed with itself (i.e. when a sweet corn AZLAN is crossed withanother hybrid sweet corn AZLAN plant or when self-pollinated), or withanother, different hybrid sweet corn, in some embodiments, a “four-way”cross is produced. Such three and four-way hybrid seeds and plantsproduced by growing said three and four-way hybrid seeds are included inthe present invention. Methods for producing a three and four-way hybridsweet corn seeds comprising (a) crossing hybrid sweet corn designatedAZLAN sweet corn plant with a different sweet corn inbred line or hybridand (b) harvesting the resultant hybrid sweet corn seed are also part ofthe invention. The hybrid sweet corn seeds produced by the methodcomprising crossing hybrid sweet corn designated AZLAN sweet corn plantwith a different sweet corn plant such as a sweet corn inbred line orhybrid, and harvesting the resultant hybrid sweet corn seed are includedin the invention, as are included the hybrid sweet corn plant or partsthereof and seeds produced by said grown hybrid sweet corn plants.

Further included in the invention are methods for producing sweet cornseeds and plants made thereof. In some embodiments, the methods compriseself-pollinating the hybrid sweet corn designated AZLAN and harvestingthe resultant hybrid seeds. Sweet corn seeds produced by such method arealso part of the invention.

In another embodiment, this invention relates to methods for producing ahybrid sweet corn designated AZLAN from a collection of seeds.

In some embodiments, the collection contains both seeds of inbred parentline(s) of hybrid sweet corn designated AZLAN seeds and hybrid seeds ofAZLAN. Such a collection of seeds might be a commercial bag of seeds. Insome embodiments, said methods comprise planting the collection ofseeds. When planted, the collection of seeds will produce inbred parentlines of hybrid sweet corn AZLAN and hybrid plants from the hybrid seedsof AZLAN. In some embodiments, said inbred parent lines of hybrid sweetcorn designated AZLAN plants are identified as having a decreased vigorcompared to the other plants (i.e. hybrid plants) grown from thecollection of seeds. In some embodiments, said decreased vigor is due tothe inbreeding depression effect and can be identified for example by aless vigorous appearance for vegetative and/or reproductivecharacteristics including a shorter plant height, small ear size, earand kernel shape, kernel color or other characteristics. In someembodiments, seeds of the inbred parent lines of the hybrid sweet cornAZLAN are collected and, if new inbred parent plants thereof are grownand crossed in a controlled manner with each other, the hybrid sweetcorn AZLAN will be recreated.

This invention also relates to methods for producing other sweet cornplants derived from hybrid sweet corn AZLAN and to the sweet corn plantsderived by the use of methods described herein.

In some embodiments, such methods for producing a sweet corn plantderived from hybrid sweet corn AZLAN comprise (a) self-pollinating thehybrid sweet corn AZLAN plant at least once to produce a progeny plantderived from the hybrid sweet corn AZLAN. In some embodiments, themethods further comprise (b) crossing the progeny plant derived from thehybrid sweet corn AZLAN with itself or a second sweet corn plant toproduce a seed of a progeny plant of a subsequent generation. In someembodiments, the methods further comprise (c) growing the progeny plantof the subsequent generation. In some embodiments, the methods furthercomprise (d) crossing the progeny plant of the subsequent generationwith itself or a second sweet corn plant to produce a sweet corn plantfurther derived from the hybrid sweet corn AZLAN. In furtherembodiments, steps (b), step (c) and/or step (d) are repeated for atleast 1, 2, 3, 4, 5, 6, 7, 8, or more generations to produce a sweetcorn plant derived from the hybrid sweet corn AZLAN. In someembodiments, within each crossing cycle, the second plant is the sameplant as the second plant in the last crossing cycle. In someembodiments, within each crossing cycle, the second plant is differentfrom the second plant in the last crossing cycle.

Another method for producing a sweet corn plant derived from hybridsweet corn AZLAN, comprises (a) crossing the hybrid sweet corn AZLANplant with a second sweet corn plant to produce a progeny plant derivedfrom the hybrid sweet corn AZLAN. In some embodiments, the methodfurther comprises (b) crossing the progeny plant derived from the hybridsweet corn AZLAN with itself or a second sweet corn plant to produce aseed of a progeny plant of a subsequent generation. In some embodiments,the method further comprises (c) growing the progeny plant of thesubsequent generation. In some embodiments, the method further comprises(d) crossing the progeny plant of the subsequent generation with itselfor a second sweet corn plant to produce a sweet corn plant derived fromthe hybrid sweet corn AZLAN. In a further embodiment, steps (b), (c)and/or (d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, or moregenerations to produce a sweet corn plant derived from the hybrid sweetcorn AZLAN. In some embodiments, within each crossing cycle, the secondplant is the same plant as the second plant in the last crossing cycle.In some embodiments, within each crossing cycle, the second plant isdifferent from the second plant in the last crossing cycle.

More specifically, the invention comprises methods for producing a malesterile sweet corn plant, an herbicide resistant sweet corn plant, aninsect resistant sweet corn plant, a disease resistant sweet corn plant,a water-stress-tolerant plant, a heat stress tolerant plants, animproved shelf life sweet corn plant, a sweet corn plant with increasedsweetness and flavor, a sweet corn plant with increased sugar content, asweet corn plant with enhanced nutritional quality, a sweet corn plantwith improved nutritional use efficiency, a sweet corn plant withdelayed senescence or controlled ripening and/or plants with improvedsalt tolerance. In some embodiments, said methods comprise transformingthe hybrid designated AZLAN sweet corn plant with nucleic acid moleculesthat confer male sterility, herbicide resistance, insect resistance,disease resistance, water-stress tolerance, heat stress tolerance,increased shelf life, increased sweetness and flavor, enhancednutritional quality, improved nutritional use efficiency, increasedsugar content, delayed senescence or controlled ripening and/or improvedsalt tolerance, respectively. The transformed sweet corn plants or partsthereof, obtained from the provided methods, including for example amale sterile sweet corn plant, an herbicide resistant sweet corn plant,an insect resistant sweet corn plant, a disease resistant sweet cornplant, a sweet corn with water stress tolerance, a sweet corn plant withheat stress tolerance, a sweet corn plant with increased sweetness andflavor, a sweet corn plant with increased sugar content, a sweet cornwith enhanced nutritional quality, a sweet corn plant with improvednutritional use efficiency, a sweet corn plant with delayed senescenceor controlled ripening or a sweet corn plant with improved salttolerance are included in the present invention. Plants may display oneor more of the above listed traits. For the present invention and theskilled artisan, disease is understood to include, but not limited tofungal diseases, viral diseases, bacterial diseases, mycoplasm diseases,or other plant pathogenic diseases and a disease resistant plant willencompass a plant resistant to fungal, viral, bacterial, mycoplasm, andother plant pathogens.

In one aspect, the present invention provides methods of introducing asingle locus conversion conferring one or more desired trait(s) into thehybrid sweet corn AZLAN, and plants, ears and/or seeds obtained fromsuch methods. In another aspect, the present invention provides methodsof modifying a single locus conversion conferring one or more desiredtrait(s) into the hybrid sweet corn AZLAN, and plants, ears and/or seedsobtained from such methods. The desired trait(s) may be, but notexclusively, a single gene. In some embodiments, the gene is a dominantallele. In some embodiments, the gene is a partially dominant allele. Insome embodiments, the gene is a recessive allele. In some embodiments,the gene or genes will confer such traits, including but not limited tomale sterility, herbicide resistance, insect resistance, resistance forbacterial, fungal, mycoplasma or viral disease, enhanced plant qualitysuch as improved drought or salt tolerance, water-stress tolerance,improved standability, enhanced plant vigor, improved shelf life,delayed senescence or controlled ripening, enhanced nutritional qualitysuch as increased sugar content or increased sweetness, increasedtexture, flavor and aroma, improved husk length, protection for color,ear shape, kernel shape, uniformity, length or diameter, kernel color,refinement or depth, lodging resistance, yield and recovery, improvedfresh cut application, specific aromatic compounds, specific volatiles,flesh texture and specific nutritional components. For the presentinvention and the skilled artisan, disease is understood to include, butnot limited to fungal diseases, viral diseases, bacterial diseases,mycoplasma diseases, or other plant pathogenic diseases and a diseaseresistant plant will encompass a plant resistant to fungal, viral,bacterial, mycoplasma, and other plant pathogens. In one aspect, thegene or genes may be naturally occurring sweet corn gene(s) and/orspontaneous or induced mutations(s). In another aspect, genes aremutated, modified, genetically engineered through the use of NewBreeding Techniques described herein. In some embodiments, the methodfor introducing the desired trait(s) is a backcrossing process by makinguse of a series of backcrosses to at least one of the parent lines ofhybrid sweet corn designated AZLAN (a.k.a. hybrid sweet corn AZLAN orsweet corn hybrid AZLAN) during which the desired trait(s) is maintainedby selection. At least one of the parent lines of hybrid sweet corndesignated AZLAN possesses the desired trait(s) by the backcrossingprocess, and the desired trait(s) is inherited by the hybrid sweet cornprogeny plants by conventional breeding techniques known to breeders ofordinary skill in the art. The single gene converted plants or singlelocus converted plants that can be obtained by the methods are includedin the present invention.

When dealing with a gene that has been modified, for example through NewBreeding Techniques, the trait (genetic modification) could be directlymodified into the newly developed hybrid sweet corn plant and/or atleast one of the parent lines of hybrid sweet corn AZLAN. Alternatively,if the trait is not modified into each newly developed hybrid sweet cornplant and/or at least one of the parent lines of hybrid sweet cornAZLAN, another typical method used by breeders of ordinary skill in theart to incorporate the modified gene is to take a line already carryingthe modified gene and to use such line as a donor line to transfer themodified gene into the newly developed hybrid sweet corn plant and/or atleast one of the parent lines of the newly developed hybrid. The samewould apply for a naturally occurring trait or one arising fromspontaneous or induced mutations.

In some embodiments, the backcross breeding process of hybrid sweet cornAZLAN comprises (a) crossing one of the parental inbred line plants ofhybrid sweet corn AZLAN with plants of another line that comprise thedesired trait(s) to produce F1 progeny plants. In some embodiments, theprocess further comprises (b) selecting the F1 progeny plants that havethe desired trait(s). In some embodiments, the process further comprises(c) crossing the selected F1 progeny plants with the parental inbredsweet corn lines of hybrid sweet corn AZLAN plants to produce backcrossprogeny plants. In some embodiments, the process further comprises (d)selecting for backcross progeny plants that have the desired trait(s)and essentially all the physiological and morphological characteristicsof the sweet corn parental inbred line of hybrid sweet corn AZLAN toproduce selected backcross progeny plants. In some embodiments, theprocess further comprises (e) repeating steps (c) and (d) one, two,three, four, five six, seven, eight, nine or more times in succession toproduce selected, second, third, fourth, fifth, sixth, seventh, eighth,ninth or higher backcross progeny plants that have the desired trait(s)and essentially all the characteristics of the parental inbred sweetcorn line of hybrid sweet corn AZLAN, and/or have the desired trait(s)and essentially all the physiological and morphological characteristicsof the parental sweet corn inbred line of hybrid sweet corn AZLAN,and/or have the desired trait(s) and otherwise essentially all thephysiological and morphological characteristics of the parental inbredsweet corn line of sweet corn hybrid AZLAN, including but not limited towhen grown in the same environmental conditions or including but notlimited to at a 5% significance level when grown in the sameenvironmental conditions. The sweet corn plants or seed produced by themethods are also part of the invention, as are the hybrid sweet cornAZLAN plants that comprised the desired trait. Backcrossing breedingmethods, well known to one skilled in the art of plant breeding will befurther developed in subsequent parts of the specification.

An embodiment of this invention is a method of making a backcrossconversion of hybrid sweet corn AZLAN. In some embodiments, the methodcomprises crossing one of the parental sweet corn inbred line plants ofhybrid sweet corn AZLAN with a donor plant comprising a mutant gene(s),a naturally occurring gene(s) or a gene(s) and/or sequence(s) modifiedthrough New Breeding Techniques conferring one or more desired traits toproduce F1 progeny plants. In some embodiments, the method furthercomprises selecting an F1 progeny plant comprising the naturallyoccurring gene(s), mutant gene(s) or gene(s) and/or sequences(s)modified through New Breeding Techniques conferring the one or moredesired traits. In some embodiments, the method further comprisesbackcrossing the selected progeny plant to the parental sweet corninbred line plants of hybrid sweet corn AZLAN. This method may furthercomprise the step of obtaining a molecular marker profile of theparental sweet corn inbred line plants of hybrid sweet corn AZLAN andusing the molecular marker profile to select for the progeny plant withthe desired trait and the molecular marker profile of the parental sweetcorn inbred line plants of hybrid sweet corn AZLAN. In some embodiments,this method further comprises crossing the backcross progeny plant AZLANof the parental sweet corn inbred line plant of hybrid sweet corn AZLANcontaining the naturally occurring gene(s), the mutant gene(s) or themodified gene(s) and or sequences modified through New BreedingTechniques conferring the one or more desired trait with the secondparental inbred sweet corn line plants of hybrid sweet corn AZLAN inorder to produce the hybrid sweet corn AZLAN comprising the naturallyoccurring gene(s), the mutant gene(s) or modified gene(s) and/orsequences modified through New Breeding Techniques conferring the one ormore desired traits. The plants or parts thereof produced by suchmethods are also part of the present invention.

In some embodiments of the invention, the number of loci that may betransferred and/or backcrossed into the parental sweet corn inbred lineof hybrid sweet corn AZLAN is at least 1, 2, 3, 4, 5, or more.

A single locus may contain several genes. A single locus conversion alsoallows for making one or more site specific changes to the plant genome,such as, without limitation, one or more nucleotide changes, deletions,insertions, substitutions, etc. In some embodiments, the single locusconversion is performed by genome editing, a.k.a. genome editing withengineered nucleases (GEEN). In some embodiments, the genome editingcomprises using one or more engineered nucleases. In some embodiments,the engineered nucleases include, but are not limited to Zinc fingernucleases (ZFNs), Transcription Activator-Like Effector Nucleases(TALENs), the CRISPR/Cas system, engineered meganuclease, engineeredhoming endonucleases and endonucleases for DNA guided genome editing(Gao et al., Nature Biotechnology (2016), doi: 10.1038/nbt.3547). Insome embodiments, the single locus conversion changes one or severalnucleotides of the plant genome. Such genome editing techniques are someof the techniques now known by the person skilled in the art and hereinare collectively referred to as “New Breeding Techniques”. In someembodiments, one or more above-mentioned genome editing method isdirectly applied on a plant of the present invention, rather than on theparental sweet corn inbred lines of hybrid sweet corn AZLAN.Accordingly, a cell containing edited genome, or a plant part containingsuch cell can be isolated and used to regenerate a novel plant which hasa new trait conferred by said genome editing, and essentially all thephysiological and morphological characteristics of hybrid sweet cornplant AZLAN.

The invention further provides methods for developing sweet corn plantsin a sweet corn plant breeding program using plant breeding techniquesincluding but not limited to, recurrent selection, backcrossing,pedigree breeding, genomic selection, molecular marker (IsozymeElectrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reactions (AP-PCRs), DNA Amplification Fingerprintings(DAFs), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, Single NucleotidePolymorphisms (SNPs), enhanced selection, genetic markers, enhancedselection and transformation. Seeds, sweet corn plants, and partsthereof produced by such breeding methods are also part of theinvention.

The invention also relates to variants, mutants and trivialmodifications of the seed or plant of the hybrid sweet corn AZLAN orinbred parental lines thereof. Variants, mutants and trivialmodifications of the seed or plant of hybrid sweet corn AZLAN or inbredparental lines thereof can be generated by methods available to oneskilled in the art, including but not limited to, mutagenesis (e.g.,chemical mutagenesis, radiation mutagenesis, transposon mutagenesis,insertional mutagenesis, signature tagged mutagenesis, site-directedmutagenesis, and natural mutagenesis), knock-outs/knock-ins, antisenseoligonucleotides, RNA interference and other techniques such as the NewBreeding Techniques. For more information of mutagenesis in plants, suchas agents or protocols, see Acquaah et al. (Principles of plant geneticsand breeding, Wiley-Blackwell, 2007, ISBN 1405136464, 9781405136464,which is herein incorporated by reference in its entity).

The invention also relates to a mutagenized population of the hybridsweet corn AZLAN and methods of using such populations. In someembodiments, the mutagenized population can be used in screening for newsweet corn plants which comprise essentially one or more of or all themorphological and physiological characteristics of hybrid sweet cornAZLAN. In some embodiments, the new sweet corn plants obtained from thescreening process comprise essentially all the morphological andphysiological characteristics of the hybrid sweet corn AZLAN, and one ormore additional or different morphological and physiologicalcharacteristics that the hybrid sweet corn AZLAN does not have.

This invention also is directed to methods for producing a sweet cornplant by crossing a first parent sweet corn plant with a second parentsweet corn plant, wherein either the first or second parent sweet cornplant is a hybrid sweet corn plant of AZLAN. Further, both first andsecond parent sweet corn plants can come from the hybrid sweet cornplant AZLAN. Further, the hybrid sweet corn plant AZLAN can beself-pollinated i.e. the pollen of a hybrid sweet corn plant AZLAN canpollinate the ovule of the same hybrid sweet corn plant AZLAN. Whencrossed with another sweet corn plant, a hybrid seed is produced. Suchmethods of hybridization and self-pollination are well known to thoseskilled in the art of breeding.

An inbred sweet corn line such as one of the parental lines of hybridsweet corn AZLAN has been produced through several cycles ofself-pollination and is therefore to be considered as a homozygous line.An inbred line can also be produced though the dihaploid system whichinvolves doubling the chromosomes from a haploid plant or embryo thusresulting in an inbred line that is genetically stable (homozygous) andcan be reproduced without altering the inbred line. Haploid plants couldbe obtained from haploid embryos that might be produced frommicrospores, pollen, anther cultures or ovary cultures or spontaneoushaploidy. The haploid embryos may then be doubled by chemical treatmentssuch as by colchicine or be doubled autonomously. The haploid embryosmay also be grown into haploid plants and treated to induce thechromosome doubling. In either case, fertile homozygous plants areobtained. A hybrid variety is classically created through thefertilization of an ovule from an inbred parental line by the pollen ofanother, different inbred parental line. Due to the homozygous state ofthe inbred line, the produced gametes carry a copy of each parentalchromosome. As both the ovule and the pollen bring a copy of thearrangement and organization of the genes present in the parental lines,the genome of each parental line is present in the resulting F1 hybrid,theoretically in the arrangement and organization created by the plantbreeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross shall be stable. The F1 hybrid is then acombination of phenotypic characteristics issued from two arrangementand organization of genes, both created by a person skilled in the artthrough the breeding process.

Still further, this invention is also directed to methods for producinga sweet corn plant derived from hybrid sweet corn AZLAN by crossinghybrid sweet corn plant AZLAN with a second sweet corn plant. In someembodiments, the methods further comprise obtaining a progeny seed fromthe cross. In some embodiments, the methods further comprise growing theprogeny seed, and possibly repeating the crossing and growing steps withthe hybrid sweet corn plant AZLAN derived plant from 0 to 7 or moretimes. Thus, any such methods using the hybrid sweet corn plant AZLANare part of this invention: selfing, backcrosses, hybrid production,crosses to populations, and the like. All plants produced using hybridsweet corn plant AZLAN as a parent are within the scope of thisinvention, including plants derived from hybrid sweet corn plant AZLAN.In some embodiments, such plants have one, more than one or all thephysiological and morphological characteristics of the hybrid sweet cornplant AZLAN listed in Table 1 including but not limited to as determinedat the 5% significance level when grown in the same environmentalconditions. In some embodiments, such plants might exhibit additionaland desired characteristics or traits such as high seed yield, high seedgermination, seedling vigor, early maturity, high yield, diseasetolerance or resistance, lodging resistance, and adaptability for soiland climate conditions. Consumer-driven traits, such as a preference fora given ear size, kernel color, kernel texture, kernel taste, kernelfirmness, kernel sugar content are other traits that may be incorporatedinto new sweet corn plants developed by this invention.

A sweet corn plant can also be propagated vegetatively. A part of theplant, for example a shoot tissue, is collected, and a new plant isobtained from the part. Such part typically comprises an apical meristemof the plant. The collected part is transferred to a medium allowingdevelopment of a plantlet, including for example rooting or developmentof shoots, or is grafted onto a sweet corn plant or a rootstock preparedto support growth of shoot tissue. This is achieved using methods wellknown in the art. Accordingly, in one embodiment, a method ofvegetatively propagating a plant of the present invention comprisescollecting a part of a plant according to the present invention, e.g. ashoot tissue, and obtaining a plantlet from said part. In oneembodiment, a method of vegetatively propagating a plant of the presentinvention comprises: (a) collecting tissue of a plant of the presentinvention; (b) rooting said proliferated shoots to obtain rootedplantlets. In one embodiment, a method of vegetatively propagating aplant of the present invention comprises: (a) collecting tissue of aplant of the present invention; (b) cultivating said tissue to obtainproliferated shoots; (c) rooting said proliferated shoots to obtainrooted plantlets. In one embodiment, such method further comprisesgrowing a plant from said plantlets. In one embodiment, an ear isharvested from said plant. In one embodiments, such ear, kernels andplants have all the physiological and morphological characteristics ofear, kernels and plants of hybrid sweet corn designated AZLAN when grownin the same environmental conditions. In one embodiment, the ear and/orits kernels is processed into products such as canned sweet corn kernelsand/or parts thereof, freeze dried or frozen kernel and/or partsthereof, fresh or prepared ear or kernels and parts thereof or pastes,powders, sauces, purees and the like.

The invention is also directed to the use of the hybrid sweet corn plantAZLAN in a grafting process. In one embodiment, the hybrid sweet cornplant AZLAN is used as the scion while in another embodiment, the hybridsweet corn plant AZLAN is used as a rootstock.

In some embodiments, the present invention teaches a seed of hybridsweet corn designated AZLAN, wherein a representative sample of seed ofsaid hybrid is deposited under NCIMB No. 43888.

In some embodiments, the present invention teaches a sweet corn plant,or a part thereof, produced by growing the deposited AZLAN seed.

In some embodiments, the present invention teaches a sweet corn plantpart, wherein the sweet corn part is selected from the group consistingof: a leaf, a flower, a kernel, an ear, a stalk, a root, a rootstock, aseed, an embryo, a peduncle, a stamen, an anther, a pistil, an ovule, apollen, a cell, a rootstock, and a scion.

In some embodiments, the present invention teaches a sweet corn plant,or a part thereof, having all the characteristics of hybrid sweet cornAZLAN as listed in Table 1 of this invention including but not limitedto as determined at the 5% significance level when grown in the sameenvironmental conditions.

In some embodiments, the present invention teaches a sweet corn plant,or a part thereof, having all the physiological and morphologicalcharacteristics of hybrid sweet corn AZLAN, wherein a representativesample of seed of said hybrid was deposited under NCIMB No. 43888.

In some embodiments, the present invention teaches a tissue culture ofregenerable cells produced from the plant or part grown from thedeposited AZLAN seed, wherein cells of the tissue culture are producedfrom a plant part selected from the group consisting of protoplasts,embryos, meristematic cells, callus, pollens, ovules, flowers, seeds,leaves, roots, root tips, anthers, stems, petioles, fruits, axillarybuds, cotyledons and hypocotyls. In some embodiments, the plant partincludes protoplasts produced from a plant grown from the depositedAZLAN seed.

In some embodiments, the present invention teaches a compositioncomprising regenerable cells produced from the plant or part thereofgrown from the deposited hybrid AZLAN seed, or other part or cellthereof. In some embodiments, the composition comprises a growth media.In some embodiments, the growth media is solid or a syntheticcultivation medium. In some embodiments, the composition is a sweet cornplant regenerated from the tissue culture from a plant grown from thedeposited AZLAN seed, said plant having the characteristics of hybridsweet corn AZLAN, wherein a representative sample of seed of said hybridis deposited under NCIMB No. 43888.

In some embodiments, the present invention teaches a sweet corn ears andkernels produced from the plant grown from the deposited AZLAN seed.

In some embodiments, such kernels have all the physiological andmorphological characteristics of hybrid sweet corn designated AZLANkernels when grown in the same environmental conditions.

In some embodiments, methods of producing said sweet corn ear comprise(a) growing the sweet corn plant from deposited AZLAN seed to produce asweet corn ear, and (b) harvesting said sweet corn ear. In someembodiments, the present invention also teaches a sweet corn earproduced by the method of producing sweet corn ears and/or kernels asdescribed above. In some embodiments, such ear and kernels have all thephysiological and morphological characteristics of ear and kernels ofhybrid sweet corn designated AZLAN (e.g. those listed in Table 1) whengrown in the same environmental conditions.

In some embodiments, the present invention teaches methods for producinga sweet corn seed comprising crossing a first parent sweet corn plantwith a second parent sweet corn plant and harvesting the resultant sweetcorn seed, wherein said first parent sweet corn plant and/or secondparent sweet corn plant is the sweet corn plant produced from thedeposited AZLAN seed or a sweet corn plant having all thecharacteristics of hybrid sweet corn AZLAN as listed in Table 1including but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions.

In some embodiments, the present invention teaches methods for producinga sweet corn seed comprising self-pollinating the sweet corn plant grownfrom the deposited AZLAN seed and harvesting the resultant sweet cornseed.

In some embodiments, the present invention teaches the seed produced byany of the above described methods.

In some embodiments, the present invention teaches methods ofvegetatively propagating the sweet corn plant grown from the depositedAZLAN seed, said method comprising collecting a part of a plant grownfrom the deposited AZLAN seed and regenerating a plant from said part.

In some embodiments, the method further comprises harvesting an earand/or kernels and/or seeds from said vegetatively propagated plant. Insome embodiments, the method further comprises harvesting a kernel fromsaid vegetatively propagated plant.

In some embodiments, the present invention teaches the plant and the earand/or kernels and/or seeds of plants vegetatively propagated from partsof plants grown from the deposited AZLAN seed. In some embodiments, suchplant, ear and/or kernels and/or seeds have all the physiological andmorphological characteristics of AZLAN plant, ear and/or kernels and/orseeds of hybrid sweet corn AZLAN (e.g. those listed in Table 1) whengrown in the same environmental conditions.

In some embodiments, the present invention teaches methods of producinga sweet corn plant derived from the hybrid sweet corn AZLAN. In someembodiment, the methods comprise (a) self-pollinating the plant grownfrom the deposited AZLAN seed at least once to produce a progeny plantderived from sweet corn hybrid AZLAN. In some embodiments, the methodfurther comprises (b) crossing the progeny plant derived from sweet cornhybrid AZLAN with itself or a second sweet corn plant to produce a seedof a progeny plant of a subsequent generation; and; (c) growing theprogeny plant of the subsequent generation from the seed, and (d)crossing the progeny plant of the subsequent generation with itself or asecond sweet corn plant to produce a sweet corn plant derived from thehybrid sweet corn variety AZLAN. In some embodiments said methodsfurther comprise the step of: (e) repeating steps (b), (c) and/or (d)for at least 1, 2, 3, 4, 5, 6, 7, or more generation to produce a sweetcorn plant derived from the hybrid sweet corn variety AZLAN.

In some embodiments, the present invention teaches methods of producinga sweet corn plant derived from the hybrid sweet corn AZLAN, the methodscomprising (a) crossing the plant grown from the deposited AZLAN seedwith a second sweet corn plant to produce a progeny plant derived fromhybrid sweet corn AZLAN. In some embodiments, the method furthercomprises; (b) crossing the progeny plant derived from hybrid sweet cornAZLAN with itself or a second sweet corn plant to produce a seed of aprogeny plant of a subsequent generation; and; (c) growing the progenyplant of the subsequent generation from the seed; (d) crossing theprogeny plant of the subsequent generation with itself or a second sweetcorn plant to produce a sweet corn plant derived from the hybrid sweetcorn variety AZLAN. In some embodiments said methods further comprisethe steps of: (e) repeating steps (b), (c) and/or (d) for at least 1, 2,3, 4, 5, 6, 7 or more generations to produce a sweet corn plant derivedfrom the hybrid sweet corn variety AZLAN.

In some embodiments, the present invention teaches plants grown from thedeposited AZLAN seed wherein said plants comprise a single locusconversion. As used herein, the term “a” or “an” refers to one or moreof that entity; for example, “a single locus conversion” refers to oneor more single locus conversions or at least one single locusconversion. As such, the terms “a” (or “an”), “one or more” and “atleast one” are used interchangeably herein. In addition, reference to“an element” by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the elements are present, unless thecontext clearly requires that there is one and only one of the elements.

In some embodiments, the present invention teaches a method of producinga plant of hybrid sweet corn designated AZLAN comprising at least onedesired trait, the method comprising introducing a single locusconversion conferring the desired trait into hybrid sweet corndesignated AZLAN, whereby a plant of hybrid sweet corn designated AZLANcomprising the desired trait is produced.

In some embodiments, the present invention teaches a sweet corn plant,comprising a single locus conversion and essentially all thecharacteristics of hybrid sweet corn designated AZLAN listed in Table 1when grown under the same environmental conditions, wherein arepresentative sample of seed of said hybrid has been deposited underNCIMB No. 43888. In other embodiments, the single locus conversion isintroduced into the plant by the use of recurrent selection, mutationbreeding, wherein said mutation breeding selects for a mutation that isspontaneous or artificially induced, backcrossing, pedigree breeding,haploid/double haploid production, marker-assisted selection, genetictransformation, genomic selection, Zinc finger nuclease (ZFN)technology, oligonucleotide directed mutagenesis, cisgenesis,intragenesis, RNA-dependent DNA methylation, agro-infiltration,Transcription Activation-Like Effector Nuclease (TALENs), CRISPR/Cassystem, engineered meganuclease, engineered homing endonuclease, and DNAguided genome editing.

In some embodiments, the plant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more single locus conversions. In some embodiments, the plantcomprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single locusconversions, but essentially all the other physiological andmorphological characteristics of hybrid sweet corn plant AZLAN listed inTable 1. In some embodiments, the plant comprises at least one singlelocus conversion and essentially all the physiological and morphologicalcharacteristics of hybrid sweet corn plant AZLAN listed in Table 1. Inother embodiments, the plant comprises one single locus conversion andessentially all of the other physiological and morphologicalcharacteristics of hybrid sweet corn plant AZLAN listed in Table 1.

In some embodiments said single locus conversion confers said plantswith a trait selected from the group consisting of male sterility, malefertility, herbicide resistance, insect resistance, resistance forbacterial, fungal, mycoplasma or viral disease, enhanced plant qualitysuch as improved drought or salt tolerance, water stress tolerance,improved standability, enhanced plant vigor, improved shelf life,delayed senescence or controlled ripening, increased nutritional qualitysuch as increased sugar content or increased sweetness, increasedtexture, flavor and aroma, improved ear length and/or size, protectionfor color, ear shape, kernel shape, uniformity, length or diameter,refinement or depth lodging resistance, yield and recovery when comparedto a suitable check/comparison plant. In further embodiments, the singlelocus conversion confers said plant with herbicide resistance.

In some embodiments, the check plant is a hybrid sweet corn AZLAN nothaving said single locus conversion conferring the desired trait(s). Insome embodiments, at least one single locus conversion is anartificially mutated gene or a gene or nucleotide sequence modifiedthrough the use of New Breeding Techniques.

In some embodiments, the present invention teaches methods of producinga sweet corn plant, comprising grafting a rootstock or a scion of thehybrid sweet corn plant grown from the deposited AZLAN seed to anothersweet corn plant. In some embodiments, the present invention teachesmethods for producing nucleic acids, comprising isolating nucleic acidsfrom the plant grown from the deposited AZLAN seed, or a part, or a cellthereof. In some embodiments, the present invention teaches methods forproducing a second sweet corn plant, comprising applying plant breedingtechniques to the plant grown from the deposited AZLAN seed, or partthereof to produce the second sweet corn plant.

In some embodiments, the present invention provides a method ofproducing a commodity plant product comprising collecting the commodityplant product from the plant of the present invention. The commodityplant product produced by said method is also part of the presentinvention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Adaptability: A plant that has adaptability is a plant able to grow wellin different growing conditions (climate, soils, etc.).

Allele: An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Androecious plant: A plant having staminate flowers only.

Backcrossing: Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, afirst-generation hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Commodity plant product: A “commodity plant product” refers to anycomposition or product that is comprised of material derived from aplant, seed, plant cell, or plant part of the present invention.Commodity plant products may be sold to consumers and can be viable ornonviable. Nonviable commodity products include but are not limited tononviable seeds and grains; processed seeds, seed parts, and plantparts; dehydrated plant tissue, frozen plant tissue, and processed planttissue; seeds and plant parts processed for animal feed for terrestrialand/or aquatic animal consumption, oil, meal, flour, flakes, bran,fiber, paper, tea, coffee, silage, crushed of whole grain, and any otherfood for human or animal consumption; biomasses and fuel products; andraw material in industry.

Collection of seeds: In the context of the present invention acollection of seeds is a grouping of seeds mainly containing similarkind of seeds, for example hybrid seeds having the inbred line of theinvention as a parental line, but that may also contain, mixed togetherwith this first kind of seeds, a second, different kind of seeds, of oneof the inbred parent lines, for example the inbred line of the presentinvention. A commercial bag of hybrid seeds having the inbred line ofthe invention as a parental line and containing also the inbred lineseeds of the invention would be, for example such a collection of seeds.

Daily heat unit value. The daily heat unit value is calculated asfollows: (the maximum daily temperature+the minimum daily temperature)/2minus 50. All temperatures are in degrees Fahrenheit. The maximumtemperature threshold is 86 degrees, if temperatures exceed this, 86 isused. The minimum temperature threshold is 50 degrees, if temperaturesgo below this, 50 is used.

Decreased vigor: A plant having a decreased vigor in the presentinvention is a plant that, compared to other plants has a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small ear size, ear and kernel shape, ear color,fewer kernel or other characteristics.

Endosperm Type: Endosperm type refers to endosperm genes and otherquality affecting modifiers and types such as starch, sugary alleles(su1, su2, etc.), sugary enhancer (se1) or extender, waxy, amyloseextender, dull, brittle alleles (bt1, bt2, etc.) shrunken-2 (sh2) andother sh alleles, and any combination of these.

Easy to pick ears: An ear that is easy to pick is an ear that easilydetaches from the plant. Once grabbed and twisted, the ear will breakbetween the peduncle and the stem. For ears not easy to pick, thepeduncle breaks off the ear. An ear that is easy to pick is also an earthat is easily accessible for harvest. When plants have an open planthabit, the ears are harvested more easily than when the plants haveclosed habit.

Enhanced nutritional quality: The nutritional quality of the sweet cornof the present invention can be enhanced by the introduction of severaltraits comprising a higher endosperm sugar content, increased sweetness,a thinner pericarp, various endosperm types or mutants. Examples ofgenes governing such traits are the “sugary” gene (su1), the “SugaryEnhancer” gene (se1) and the “Shrunken-2” gene (sh2).

Essentially all the physiological and morphological characteristics: Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having all the physiological andmorphological characteristics of a plant of the present invention,except for desired characteristic(s), which can be indirectly obtainedfrom another plant possessing at least one single locus conversion via aconventional breeding program (such as backcross breeding) or directlyobtained by introduction of at least one single locus conversion via NewBreeding Techniques. In some embodiments, one of the non-limitingexamples for a plant having (and/or comprising) essentially all thephysiological and morphological characteristics shall be a plant havingall the physiological and morphological characteristics of a plant ofthe present invention other than desired, additionaltrait(s)/characteristic(s) conferred by a single locus conversionincluding, but not limited to, a converted or modified gene.

Field holding ability: Field holding ability is the ability for kernelquality to maintain even after kernel is ripe.

Grafting: Grafting is the operation by which a rootstock is grafted witha scion. The primary motive for grafting is to avoid damages bysoil-born pest and pathogens when genetic or chemical approaches fordisease management are not available. Grafting a susceptible scion ontoa resistant rootstock can provide a resistant cultivar without the needto breed the resistance into the cultivar. In addition, grafting mayenhance tolerance to abiotic stress, increase yield and result in moreefficient water and nutrient uses.

HTU: HTU is the summation of the daily heat unit value calculated fromemergence to harvest.

Good Seed Producer: A plant is a good seed producer when it producesnumerous seeds. For sweet corn, a good seed producing plant will producean average of 20 grams of seeds during the harvest season.

Gynoecious plant: A plant having pistillate flowers only.

Immunity to disease(s) and or insect(s): A sweet corn plant which is notsubject to attack or infection by specific disease(s) and or insect(s)is considered immune.

Industrial usage: The industrial usage of the sweet corn of the presentinvention comprises the use of the sweet corn ears or kernels forconsumption, whether as fresh products or in canning, freezing or anyother industries.

Intermediate resistance to disease(s) and or insect(s): A sweet cornplant that restricts the growth and development of specific disease(s)and or insect(s), but may exhibit a greater range of symptoms or damagecompared to a resistant plant. Intermediate resistant plants willusually show less severe symptoms or damage than susceptible plantvarieties when grown under similar environmental conditions and/orspecific disease(s) and or insect(s) pressure, but may have heavy damageunder heavy pressure. Intermediate resistant sweet corn plants are notimmune to the disease(s) and or insect(s).

Maturity: In the region of best adaptability, maturity is the number ofdays from seeding to harvesting.

New Breeding Techniques: New breeding techniques (NBTs) are said ofvarious new technologies developed and/or used to create newcharacteristics in plants through genetic variation, the aim beingtargeted mutagenesis, targeted introduction of new genes or genesilencing (RdDM). The following breeding techniques are within the scopeof NBTs: targeted sequence changes facilitated through the use of Zincfinger nuclease (ZFN) technology (ZFN-1, ZFN-2 and ZFN-3, see U.S. Pat.No. 9,145,565, incorporated by reference in its entirety),Oligonucleotide directed mutagenesis (ODM, a.k.a., site-directedmutagenesis), Cisgenesis and intragenesis, epigenetic approaches such asRNA-dependent DNA methylation (RdDM, which does not necessarily changenucleotide sequence but can change the biological activity of thesequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration for transient gene expression (agro-infiltration“sensu stricto”, agro-inoculation, floral dip), TranscriptionActivator-Like Effector Nucleases (TALENs, see U.S. Pat. Nos. 8,586,363and 9,181,535, incorporated by reference in their entireties), theCRISPR/Cas system (see U.S. Pat. Nos. 8,697,359; 8,771,945; 8,795,965;8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814;8,945,839; 8,993,233; and 8,999,641, which are all hereby incorporatedby reference), engineered meganuclease, engineered homing endonucleases,DNA guided genome editing (Gao et al., Nature Biotechnology (2016), doi:10.1038/nbt.3547, incorporated by reference in its entirety), andSynthetic genomics. A major part of today's targeted genome editing,another designation for New Breeding Techniques, is the applications toinduce a DNA double strand break (DSB) at a selected location in thegenome where the modification is intended. Directed repair of the DSBallows for targeted genome editing. Such applications can be utilized togenerate mutations (e.g., targeted mutations or precise native geneediting) as well as precise insertion of genes (e.g., cisgenes,intragenes, or transgenes). The applications leading to mutations areoften identified as site-directed nuclease (SDN) technology, such asSDN1, SDN2 and SDN3. For SDN1, the outcome is a targeted, non-specificgenetic deletion mutation: the position of the DNA DSB is preciselyselected, but the DNA repair by the host cell is random and results insmall nucleotide deletions, additions or substitutions. For SDN2, a SDNis used to generate a targeted DSB and a DNA repair template (a shortDNA sequence identical to the targeted DSB DNA sequence except for oneor a few nucleotide changes) is used to repair the DSB: this results ina targeted and predetermined point mutation in the desired gene ofinterest. As to the SDN3, the SDN is used along with a DNA repairtemplate that contains new DNA sequence (e.g. gene). The outcome of thetechnology would be the integration of that DNA sequence into the plantgenome. The most likely application illustrating the use of SDN3 wouldbe the insertion of cisgenic, intragenic, or transgenic expressioncassettes at a selected genome location. A complete description of eachof these techniques can be found in the report made by the JointResearch Center (JRC) Institute for Prospective Technological Studies ofthe European Commission in 2011 and titled “New plant breedingtechniques—State-of-the-art and prospects for commercial development”,which is incorporated by reference in its entirety.

Plant adaptability: A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant cell: As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture, or incorporated in a plant or plantpart.

Plant Part: As used herein, the term “plant part”, “part thereof” or“parts thereof” includes plant cells, plant protoplasts, plant celltissue cultures from which sweet corn plants can be regenerated, plantcalli, plant clumps and plant cells that are intact in plants or partsof plants, such as embryos, pollens, ovules, flowers, seeds, kernels,rootstocks, scions, stems, roots, anthers, pistils, root tips, leaves,meristematic cells, axillary buds, hypocotyls, cotyledons, ovaries, seedcoats, endosperms and the like. In some embodiments, the plant part atleast comprises at least one cell of said plant. In some embodiments,the plant part is further defined as a pollen, a meristem, a cell or anovule. In some embodiments, a plant regenerated from the plant part hasall of the phenotypic and morphological characteristics of a sweet cornhybrid of the present invention, including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

Quantitative Trait Loci (QTL): Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration: Regeneration refers to the development of a plant fromtissue culture.

Resistance to disease(s) and or insect(s): A sweet corn plant thatrestricts the growth and development of specific disease(s) and orinsect(s) under normal disease(s) and or insect(s) attack pressure whencompared to susceptible plants. These sweet corn plants can exhibit somesymptoms or damage under heavy disease(s) and or insect(s) pressure.Resistant sweet corn plants are not immune to the disease(s) and orinsect(s).

Root Lodging: The root lodging is the percentage of plants that rootlodge; i.e., those that lean from the vertical axis at an approximate 30degree angle or greater would be counted as root lodged.

Rootstock: A rootstock is the lower part of a plant capable of receivinga scion in a grafting process.

Scion: A scion is the higher part of a plant capable of being graftedonto a rootstock in a grafting process.

Single locus converted (conversion): Single locus converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing, wherein essentially all the desired morphologicaland physiological characteristics of a plant are recovered in additionto a single locus transferred into the plant via the backcrossingtechnique or via genetic engineering. A single locus converted plant canalso be referred to a plant with a single locus conversion obtainedthough simultaneous and/or artificially induced mutagenesis or throughthe use of New Breeding Techniques described in the present invention.In some embodiments, the single locus converted plant has essentiallyall the desired morphological and physiological characteristics of theoriginal variety in addition to a single locus converted by spontaneousand/or artificially induced mutations, which is introduced and/ortransferred into the plant by the plant breeding techniques such asbackcrossing. In other embodiments, the single locus converted plant hasessentially all the desired morphological and physiologicalcharacteristics of the original variety in addition to a single locus,gene or nucleotide sequence(s) converted, mutated, modified orengineered through the New Breeding Techniques taught herein. In thepresent invention, single locus converted (conversion) can beinterchangeably referred to single gene converted (conversion).

Susceptible to disease(s) and or insect(s): A sweet corn plant that issusceptible to disease(s) and or insect(s) is defined as a sweet cornplant that has the inability to restrict the growth and development ofspecific disease(s) and or insect(s). Plants that are susceptible willshow damage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tolerance to abiotic stresses: A sweet corn plant that is tolerant toabiotic stresses has the ability to endure abiotic stress withoutserious consequences for growth, appearance and yield.

Uniformity: Uniformity, as used herein, describes the similarity betweenplants or plant characteristics which can be a described by qualitativeor quantitative measurements.

Variety: A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties ofplants). The term “variety” can be interchangeably used with “cultivar”or “hybrid in the present application”.

Yield: The yield is the tons of green corn or green weight per acre. Itcan also be defined as the number of ears per acre or per plant.

Sweet Corn Plants

Sweet corn is a particular type of maize (Zea mays, often referred to ascorn in the United States). Sweet corn naturally mutated from fieldcorn, with origins that are traced back to the Native Americans. Sweetcorn is harvested at an earlier maturity than field corn (before it isdry), for a different purpose, usually fresh produce, canning orfreezing, for human consumption, or to be eaten fresh on the cob,steamed or grilled. It has been bred therefore to be qualitatively andquantitatively different from field corn in a number of respects.

Early varieties, including those used by Native Americans, were theresult of the mutant su1 (“sugary”) allele. The sweet corn (su1)mutation causes the endosperm (storage area) of the seed to accumulateabout two times more sugar than field corn. Today many sweet corn (su1)varieties are available. They contain about 5-10% sugar by weight.

Supersweet corns are varieties of sweet corn which produce higher thannormal levels of sugar, first recognized by University of Illinois atUrbana—Champaign professor John Laughnan. He was investigating twospecific genes in sweet corn, one of which, the sh2 (shrunken2) genecaused the corn to shrivel when dry. After further investigationLaughnan discovered that the endosperm of sh2 sweet corn kernels storeless starch and from 4 to 10 times more sugar than standard sugary sweetcorn. Texture is crispy rather than creamy as with the standard andsugary enhanced varieties. Fresh market shelf life is extended due tothe ability of the kernels to retain moisture and sweetness for longerperiods of time. He published his findings in 1953, disclosing theadvantages of growing supersweet sweet corn, but many corn breederslacked enthusiasm for the new supersweet corn. Illinois Foundation SeedsInc. was the first seed company to release a supersweet corn and it wascalled Illini Xtra-Sweet, but widespread use of supersweet hybrids didnot occur until the early 1980s. The popularity of supersweet corn rosedue to its long shelf life and higher sugar content when compared toconventional sweet corn. This has improved the viability and thus thelong-distance shipping of sweet corn and has enabled manufacturers tocan sweet corn without adding extra sugar.

The third gene mutation to be discovered is the se1 or “sugary enhanced”allele. Sugary enhancer corn results in slightly increased sugar levelsand a more creamy texture due to increased levels of water solublepolysaccharides. Kernels are also generally more tender.

All of the alleles responsible for sweet corn are recessive, so it mustbe isolated from any field corn varieties that release pollen at thesame time; the endosperm develops from genes from both parents, andheterozygous kernels will be tough and starchy. The se1 and su1 allelesdo not need to be isolated from each other as the change in quality ofthe sugary enhancer hybrids will not be that dramatic. Supersweetvarieties containing the sh2 allele must be grown in isolation fromother varieties to avoid cross-pollination and resulting starchiness,either in space (various sources quote minimum isolation distances from100 to 400 feet or 30 to 120 m) or in time (i.e., the supersweet corndoes not pollinate at the same time as other corn in nearby fields).

Sweet corn hybrids come in three colors: yellow, white, and bicolorbased on whether the parental lines are yellow or white.Cross-pollination of a yellow parental line by a white parental linewill result in seed of a hybrid that when grown will produce bicolorears Cross pollination of a yellow kernel hybrid by a bicolor or whitekernel hybrid will not change the kernel color of the yellow hybrid, butmay increase the percentage of yellow kernels on the bicolor or whitekernel hybrids. This is due to the dominant nature of the allele foryellow kernel color. Although there are geographical preferences forcertain kernel colors, there is no relationship between color andsweetness.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In sweet corn these important traits include the ability of the seeds toemerge with vigor and uniformity, a strong plant type that resistslodging, a plant that carries some level of resistance for disease orinsects, a high number of fresh ears or kernels that are produced on aunit of ground, good coverage of the husk leaves over the tip of theear, easy removal of the ear from the stalk with a short shankattachment, a cylindrical ear shape and good balance between the ear'slength and diameter, kernels arranged in 14 to 20 straight rows withgood kernel depth and color, good kernel fill at the tip of the ear andgood canned, frozen and/or fresh eating quality determined by thetenderness, aroma, sweetness and flavor of the sweet corn kernels.

In some embodiments, particularly desirable traits that may beincorporated by this invention are improved resistance to differentviral, fungal, and bacterial pathogens. Important diseases include butare not limited to Northern Leaf blight (caused by Exserohilum turcicum,previously called Helminthosporium turcicum), Common Rust (caused byPuccinia sorghi), “Maize Dwarf Mosaic Virus (caused by MDMV strain A”,Sugar Cane Mosaic Virus (caused by SCMV formally known as MDMV strainB), tropical rust (caused by the fungal pathogen Physopella zeae(Mains), grey leaf spot (fungal disease associated with Cercospora spp),Goss's wilt (caused by the bacterial pathogen Clavibacter michiganensissubsp. nebraskensis (CN)), Stewart's Bacterial Wilt (caused by Pantoeastewartii), High Plains Virus (caused by HPV),” etc. Improved resistanceto insect pests is another desirable trait that may be incorporated intonew sweet corn plants developed by this invention. Insect pestsaffecting sweet corn include, but not limited to European corn borer,corn earworm, corn wireworm, Western bean cutworm fall armyworm, fleabeetles, sap beetles etc.

Sweet Corn Breeding

The goal of sweet corn breeding is to develop new, unique and superiorsweet corn inbred lines and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. Another method usedto develop new, unique and superior sweet corn inbred lines and hybridsoccurs when the breeder selects and crosses two or more parental linesfollowed by haploid induction and chromosome doubling that result in thedevelopment of dihaploid inbred lines. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations and the same is true for the utilization of thedihaploid breeding method.

During the development of new sweet corn inbreds and hybrids, the sweetcorn breeder uses sweet corn plants, but also non-commercial sweet cornplants, such as plants that may contain characteristics that the breederhas interest in having in its sweet corn inbreds and hybrids. Suchnon-commercial sweet corn plants could be regular field corn, or wildrelatives of corn such as teosinte.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred linesdeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting lines the breeder develops, exceptpossibly in a very gross and general fashion. This unpredictabilityresults in the expenditure of large research monies to develop superiornew sweet corn inbred lines and hybrids.

The development of commercial sweet corn hybrids requires thedevelopment of homozygous inbred lines, the crossing of these lines, andthe evaluation of the hybrid crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop inbred lines from breeding populations. Breeding programscombine desirable traits from two or more inbred lines or variousbroad-based sources into breeding pools from which inbred lines aredeveloped by selfing and selection of desired phenotypes or through thedihaploid breeding method followed by the selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which havecommercial potential.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

i. Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potential use asparents of new hybrid cultivars. Similarly, the development of newinbred lines through the dihaploid system requires the selection of thebest inbreds followed by two to five years of testing in hybridcombinations in replicated plots.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or more earscontaining seed from each plant in a population and blend them togetherto form a bulk seed lot. Part of the bulked seed is used to plant thenext generation and part is put in reserve. The procedure has beenreferred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than removing one seed from each ear by hand forthe single seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

ii. Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the recurrentparent and the trait of interest from the donor parent are selected andrepeatedly crossed (backcrossed) to the recurrent parent. The resultingplant is expected to have the attributes of the recurrent parent (e.g.,cultivar) and the desirable trait transferred from the donor parent.

When the term hybrid sweet corn plant is used in the context of thepresent invention, this also includes any hybrid sweet corn plant whereone or more desired trait has been introduced through backcrossingmethods, whether such trait is a naturally occurring one, a mutant, atransgenic one or a gene or a nucleotide sequence modified by the use ofNew Breeding Techniques. Backcrossing methods can be used with thepresent invention to improve or introduce one or more characteristicinto the inbred parental line, thus potentially introducing these traitsin to the hybrid sweet corn plant of the present invention. The term“backcrossing” as used herein refers to the repeated crossing of ahybrid progeny back to the recurrent parent, i.e., backcrossing one,two, three, four, five, six, seven, eight, nine, or more times to therecurrent parent. The parental sweet corn plant which contributes thegene or the genes for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental sweet corn plant to which thegene or genes from the nonrecurrent parent are transferred is known asthe recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original inbred of interest(recurrent parent) is crossed to or by a second inbred (nonrecurrentparent) that carries the gene or genes of interest to be transferred.The resulting progeny from this cross are then crossed again to or bythe recurrent parent and the process is repeated until a sweet cornplant is obtained wherein all the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, generally determined at a 5% significance levelwhen grown in the same environmental conditions, in addition to the geneor genes transferred from the nonrecurrent parent. It has to be notedthat some, one, two, three or more, self-pollination and growing ofpopulation might be included between two successive backcrosses. Indeed,an appropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non-limiting example of such a protocolwould be the following: a) the first generation F1 produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred parental line in order to find it then in the hybridmade thereof. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact, the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing, but also follow up of the characteristic(s) throughgenetically associated markers and molecular assisted breeding tools.For example, selection of progeny containing the transferred trait isdone by direct selection, visual inspection for a trait associated witha dominant allele, while the selection of progeny for a trait that istransferred via a recessive allele, such as the shrunken-2 starch mutantin sweet corn, requires selfing the progeny or using molecular markersto determine which plant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new parental inbred of a hybridsweet corn plant according to the invention but that can be improved bybackcrossing techniques. Single gene traits may or may not betransgenic. Examples of these traits include but are not limited to,male sterility (such as a cms-C, cms-T and cms-S genes), waxy starch(such as the wx gene), herbicide resistance (such as dmEPSPS gene),resistance for bacterial, rust (such as Rp1-d), fungal (such as Ht2, Ht3and HtN genes), or viral disease (gene Wsm1, Wsm2 and Wsm3 forresistance to Maize dwarf mosaic virus), insect resistance, malefertility, enhanced nutritional quality, enhanced sugar content, yieldstability and yield enhancement. These genes are generally inheritedthrough the nucleus. Some known exceptions to this are the genes formale sterility, some of which are inherited cytoplasmically, but stillact as single gene traits. Several of these single gene traits aredescribed in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

In 1981, the backcross method of breeding counted for 17% of the totalbreeding effort for inbred line development in the United States,accordingly to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,published by John Wiley & Sons, Inc., Principles of Plant Breeding). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a parental line of a hybrid variety with exactly oressentially the same adaptation, yielding ability and qualitycharacteristics of the recurrent parent but superior to that parent inthe particular characteristic(s) for which the improvement program wasundertaken. Therefore, this method provides the plant breeder with ahigh degree of genetic control of this work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because a similar variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will betheoretically modified only with regards to genes being transferred,which are maintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart wheat’ and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in California Commonalfalfa to create ‘Caliverde’. This new ‘Caliverde’ variety producedthrough the backcross process is indistinguishable from CaliforniaCommon except for its resistance to the three named diseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred or when usingmolecular markers that can identify the trait of interest.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, ‘Calady’, has been produced by Jones andDavis. As dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. ‘Lady Wright’, along grain variety was used as the donor parent and ‘Coloro’, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety ‘Calady’ was produced.

iii. Open-Pollinated Populations

The improvement of open-pollinated populations of such crops as rye,many maizes and sugar beets, herbage grasses, legumes such as alfalfaand clover, and tropical tree crops such as cacao, coconuts, oil palmand some rubber, depends essentially upon changing gene-frequenciestowards fixation of favorable alleles while maintaining a high (but farfrom maximal) degree of heterozygosity.

Uniformity in such populations is impossible and trueness-to-type in anopen-pollinated variety is a statistical feature of the population as awhole, not a characteristic of individual plants. Thus, theheterogeneity of open-pollinated populations contrasts with thehomogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement.

First, there is the situation in which a population is changed en masseby a chosen selection procedure. The outcome is an improved populationthat is indefinitely propagated by random-mating within itself inisolation.

Second, the synthetic variety attains the same end result as populationimprovement, but is not itself propagated as such; it has to bereconstructed from parental lines or clones. These plant breedingprocedures for improving open-pollinated populations are well known tothose skilled in the art and comprehensive reviews of breedingprocedures routinely used for improving cross-pollinated plants areprovided in numerous texts and articles, including: Allard, Principlesof Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principlesof Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda,Quantitative Genetics in Maize Breeding, Iowa State University Press(1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc.(1988).

A) Mass Selection

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued. In massselection, desirable individual plants are chosen, harvested, and theseed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

B) Synthetics

A synthetic variety is produced by intercrossing a number of genotypesselected for good combining ability in all possible hybrid combinations,with subsequent maintenance of the variety by open pollination. Whetherparents are (more or less inbred) seed-propagated lines, as in somesugar beet and beans (Vicia) or clones, as in herbage grasses, cloversand alfalfa, makes no difference in principle. Parents are selected ongeneral combining ability, sometimes by test crosses or toperosses, moregenerally by polycrosses. Parental seed lines may be deliberately inbred(e.g. by selfing or sib crossing). However, even if the parents are notdeliberately inbred, selection within lines during line maintenance willensure that some inbreeding occurs. Clonal parents will, of course,remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or more cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

iv. Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflower,broccoli and tomato as well as leafy vegetables such as lettuce. Hybridscan be formed in a number of different ways, including by crossing twoparents directly (single cross hybrids), by crossing a single crosshybrid with another parent (three-way or triple cross hybrids), or bycrossing two different hybrids (four-way or double cross hybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

Hybrid commercial sweet corn seed can be produced by removing thetassels from the female parent or by using a male sterile femaleincorporating manual or mechanical detasseling. Alternate strips of twosweet corn inbreds are planted in a field, and the pollen-bearingtassels are removed from one of the inbreds (female). Providing there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm. Prior to thefertilized seed. CMS plants are fertilized with pollen from anotherinbred that is not male-sterile. Pollen from the second inbred may ormay not contribute genes that make the hybrid plants male-fertile. Seedfrom detasseled fertile corn and CMS produced seed of the same hybridcan be blended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., U.S. Pat. No. 5,432,068 havedeveloped a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility, silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran anti-sense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see,Fabinjanski, et al. EPO 89/3010153.8 publication no. 329, 308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G. R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application and genotype specificallyoften limit the usefulness of the approach.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor and uniformity exhibited byF₁ hybrids is lost in the next generation (F₂). Consequently, seed fromF₂ hybrid varieties is not used for planting stock.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

v. Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., 1999, Journal of Experimental Botany, 50(337):1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to virus), and theother from the individuals having reversed phenotype (e.g., susceptibleto virus), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs, SNPs or SSRs). Markers linkedto the phenotype can be identified and used for breeding or QTL mapping.

vi. Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some embodiments the donor or recipient female parent and thedonor or recipient male parent line are planted in the same field. Theinbred male parent can be planted earlier or later than the femaleparent to ensure adequate pollen supply at the pollination time. In someembodiments, the male parent and female parent can be planted at a ratioof 1 male parent to 2-6 female parents. The male parent may be plantedat the top of the field for efficient male pollen collection duringpollination. Pollination is started when the female parent flower isready to be fertilized and there is available pollen from the maleparent. Female ear shoots that are ready to open in the following daysare identified, covered with paper cups or small paper bags that preventbee or any other insect from visiting the female flowers, and markedwith any kind of material that can be easily seen the next morning. Themale pollen of the male parent are collected after it is likely thatthere is new pollen available from that days shedding. The coveredfemale flowers of the female parent, which have ear silk showing, areun-covered and pollinated with the collected fresh male pollen of themale parent, starting as soon as the male flower sheds pollen. Thepollinated female flowers are again covered after pollination to preventcontamination by other pollen traveling by wind or by bees and any otherinsects. The pollinated female flowers are also marked. The marked earsare harvested. In some embodiments, the male pollen used forfertilization has been previously collected and stored.

vii. Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant lines. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andsweet corn.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. As DNA bases are not pairing at themismatch of the two DNA strands (the induced mutation in TILLING® or thenatural mutation or SNP in EcoTILLING), they provoke a shape change inthe double strand DNA fragment which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus; John Innes Centre (UK),focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing onArabidopsis; Southern Illinois University (USA), focusing on Soybean;John Innes Centre (UK), focusing on Lotus and Medicago; and INRA(France), focusing on Pea and Tomato.

More detailed description on methods and compositions on TILLING® can befound in U.S. Pat. No. 5,994,075, US 2004/0053236 A1, WO 2005/055704,and WO 2005/048692, each of which is hereby incorporated by referencefor all purposes.

Thus, in some embodiments, the breeding methods of the presentdisclosure include breeding with one or more TILLING plant lines withone or more identified mutations.

viii. Mutation Breeding

Mutation breeding is another method of introducing new variation andsubsequent traits into sweet corn plants. Mutations that occurspontaneously or are artificially induced can be useful sources ofvariability for a plant breeder. The goal of artificial mutagenesis isto increase the rate of mutation for a desired characteristic. Mutationrates can be increased by many different means or mutating agentsincluding temperature, long-term seed storage, tissue cultureconditions, radiation (such as X-rays, Gamma rays, neutrons, Betaradiation, or ultraviolet radiation), chemical mutagens (such as baseanalogs like 5-bromo-uracil), antibiotics, alkylating agents (such assulfur mustards, nitrogen mustards, epoxides, ethyleneamines, sulfates,sulfonates, sulfones, or lactones), azide, hydroxylamine, nitrous acidor acridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in W. R.Fehr, 1993, Principles of Cultivar Development, Macmillan Publishing Co.

New breeding techniques such as the ones involving the uses ofengineered nuclease to enhance the efficacy and precision of geneediting in combination with oligonucleotides including, but not limitedto Zinc Finger Nucleases (ZFN), TAL effector nucleases (TALENs) andclustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease Cas9 (CRISPR-Cas9) shall also be usedto generate genetic variability and introduce new traits into sweet cornvarieties.

ix. Double Haploids and Chromosome Doubling

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple backcrossing is to produce haploids and then double thechromosomes to form doubled haploids. Haploid plants can occurspontaneously, or may be artificially induced via chemical treatments orby crossing plants with inducer lines (Seymour et al. 2012, PNAS vol.109, pg. 4227-4232; Zhang et al., 2008 Plant Cell Rep. December 27(12)1851-60). The production of haploid progeny can occur via a variety ofmechanisms which can affect the distribution of chromosomes duringgamete formation. The chromosome complements of haploids sometimesdouble spontaneously to produce homozygous doubled haploids (DHs).Mixoploids, which are plants which contain cells having differentploidies, can sometimes arise and may represent plants that areundergoing chromosome doubling so as to spontaneously produce doubledhaploid tissues, organs, shoots, floral parts or plants. Another commontechnique is to induce the formation of double haploid plants with achromosome doubling treatment such as colchicine (El-Hennawy et al.,2011 Vol 56, issue 2 pg. 63-72; Doubled Haploid Production in CropPlants 2003 edited by Maluszynski ISBN 1-4020-1544-5). The production ofdoubled haploid plants yields highly uniform inbred lines and isespecially desirable as an alternative to sexual inbreeding oflonger-generation crops. By producing doubled haploid progeny, thenumber of possible gene combinations for inherited traits is moremanageable. Thus, an efficient doubled haploid technology cansignificantly reduce the time and the cost of inbred and cultivardevelopment.

x. Protoplast Fusion

In another method for breeding plants, protoplast fusion can also beused for the transfer of trait-conferring genomic material from a donorplant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell that may even be obtained with plant species that cannot beinterbred in nature is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits.

xi. Embryo Rescue

Alternatively, embryo rescue may be employed in the transfer ofresistance-conferring genomic material from a donor plant to a recipientplant. Embryo rescue can be used as a procedure to isolate embryos fromcrosses to rapidly move to the next generation of backcrossing orselfing or wherein plants fail to produce viable seed. In this process,the fertilized ovary or immature seed of a plant is tissue cultured tocreate new plants (see Pierik, 1999, In Vitro Culture of Higher Plants,Springer, ISBN 079235267X, 978-0792352679, which is incorporated hereinby reference in its entirety).

Grafting

Grafting is a process that has been used for many years in crops such ascucurbitacea, but only more recently for some commercial watermelon andtomato production. Grafting may be used to provide a certain level ofresistance to telluric pathogens such as Phytophthora or to certainnematodes. Grafting is therefore intended to prevent contact between theplant or variety to be cultivated and the infested soil. The variety ofinterest used as the graft or scion, optionally an F1 hybrid, is graftedonto the resistant plant used as the rootstock. The resistant rootstockremains healthy and provides, from the soils, the normal supply for thegraft that it isolates from the diseases. In some recent developments,it has also been shown that some rootstocks are also able to improve theagronomic value for the grafted plant and in particular the equilibriumbetween the vegetative and generative development that are alwaysdifficult to balance in sweet corn cultivation.

Breeding Evaluation

Each breeding program can include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for use as parents in new commercial cultivars; those stilldeficient in a few traits may be used as parents to produce newpopulations for further selection or in a backcross program to improvethe parent lines for a specific trait.

In some embodiments, the plants are selected on the basis of one or morephenotypic traits. Skilled persons will readily appreciate that suchtraits include any observable characteristic of the plant, including forexample growth rate, vigor, plant health, maturity, tillering,standability or tolerance to lodging, plant height, ear weight, leafarea or orientation, height, weight, color, taste, smell, sugar levels,aroma, changes in the production of one or more compounds by the plant(including for example, metabolites, proteins, drugs, carbohydrates,oils, and any other compounds).

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

It should be appreciated that in certain embodiments, plants may beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a certain feature or trait (such as a desirable featureor trait).

Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression, genotype, orpresence of genetic markers). Where the presence of one or more geneticmarker is assessed, the one or more marker may already be known and/orassociated with a particular characteristic of a plant; for example, amarker or markers may be associated with an increased growth rate ormetabolite profile. This information could be used in combination withassessment based on other characteristics in a method of the disclosureto select for a combination of different plant characteristics that maybe desirable. Such techniques may be used to identify novel quantitativetrait loci (QTLs). By way of example, plants may be selected based ongrowth rate, size (including but not limited to weight, height, leafsize, stem size, branching pattern, or the size of any part of theplant), general health, survival, tolerance to adverse physicalenvironments and/or any other characteristic, as described hereinbefore.

Further non-limiting examples include selecting plants based on: speedof seed germination; quantity of biomass produced; increased root,and/or leaf/shoot growth that leads to an increased yield (herbage orgrain or fiber or oil) or biomass production; effects on plant growththat results in an increased seed yield for a crop; effects on plantgrowth which result in an increased yield; effects on plant growth thatlead to an increased resistance or tolerance to disease includingfungal, viral or bacterial diseases, to mycoplasma, or to pests such asinsects, mites or nematodes in which damage is measured by decreasedfoliar symptoms such as the incidence of bacterial or fungal lesions, orarea of damaged foliage or reduction in the numbers of nematode cysts orgalls on plant roots, or improvements in plant yield in the presence ofsuch plant pests and diseases; effects on plant growth that lead toincreased metabolite yields; effects on plant growth that lead toimproved aesthetic appeal which may be particularly important in plantsgrown for their form, color or taste, for example the color intensity ofsweet corn exocarp (skin) of said kernel.

Molecular Breeding Evaluation Techniques

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (Transcriptome Sequencing), qRTPCR(quantitative real time PCR).

In one embodiment, the evaluating step of a plant breeding programinvolves the identification of desirable traits in progeny plants.Progeny plants can be grown in, or exposed to conditions designed toemphasize a particular trait (e.g. drought conditions for droughttolerance, lower temperatures for freezing tolerant traits). Progenyplants with the highest scores for a particular trait may be used forsubsequent breeding steps.

In some embodiments, plants selected from the evaluation step canexhibit a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120% or more improvement in aparticular plant trait compared to a control plant.

In other embodiments, the evaluating step of plant breeding comprisesone or more molecular biological tests for genes or other markers. Forexample, the molecular biological test can involve probe hybridizationand/or amplification of nucleic acid (e.g., measuring nucleic aciddensity by Northern or Southern hybridization, PCR) and/or immunologicaldetection (e.g., measuring protein density, such as precipitation andagglutination tests, ELISA (e.g., Lateral Flow test or DAS-ELISA),Western blot, immune labeling, immunosorbent electron microscopy (ISEM),and/or dot blot).

The procedure to perform a nucleic acid hybridization, an amplificationof nucleic acid (e.g., PCR, RT-PCR) or an immunological detection (e.g.,precipitation and agglutination tests, ELISA (e.g., Lateral Flow test orDAS-ELISA), Western blot, RIA, immunogold or immunofluorescent labeling,immunosorbent electron microscopy (ISEM), and/or dot blot tests) areperformed as described elsewhere herein and well-known by one skilled inthe art.

In one embodiment, the evaluating step comprises PCR (semi-quantitativeor quantitative), wherein primers are used to amplify one or morenucleic acid sequences of a desirable gene, or a nucleic acid associatedwith said gene, or QTL or a desirable trait (e.g., a co-segregatingnucleic acid, or other marker).

In another embodiment, the evaluating step comprises immunologicaldetection (e.g., precipitation and agglutination tests, ELISA (e.g.,Lateral Flow test or DAS-ELISA), Western blot, RIA, immuno labeling(gold, fluorescent, or other detectable marker), immunosorbent electronmicroscopy (ISEM), and/or dot blot), wherein one or more gene ormarker-specific antibodies are used to detect one or more desirableproteins. In one embodiment, said specific antibody is selected from thegroup consisting of polyclonal antibodies, monoclonal antibodies,antibody fragments, and combination thereof.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be utilizedin the present disclosure to determine expression of a gene to assistduring the selection step of a breeding scheme. It is a variant ofpolymerase chain reaction (PCR), a laboratory technique commonly used inmolecular biology to generate many copies of a DNA sequence, a processtermed “amplification”. In RT-PCR, however, RNA strand is first reversetranscribed into its DNA complement (complementary DNA, or cDNA) usingthe enzyme reverse transcriptase, and the resulting cDNA is amplifiedusing traditional or real-time PCR.

RT-PCR utilizes a pair of primers, which are complementary to a definedsequence on each of the two strands of the mRNA. These primers are thenextended by a DNA polymerase and a copy of the strand is made after eachcycle, leading to logarithmic amplification.

RT-PCR includes three major steps. The first step is the reversetranscription (RT) where RNA is reverse transcribed to cDNA using areverse transcriptase and primers. This step is very important in orderto allow the performance of PCR since DNA polymerase can act only on DNAtemplates. The RT step can be performed either in the same tube with PCR(one-step PCR) or in a separate one (two-step PCR) using a temperaturebetween 40° C. and 60° C., depending on the properties of the reversetranscriptase used.

The next step involves the denaturation of the dsDNA at 95° C., so thatthe two strands separate and the primers can bind again at lowertemperatures and begin a new chain reaction. Then, the temperature isdecreased until it reaches the annealing temperature which can varydepending on the set of primers used, their concentration, the probe andits concentration (if used), and the cation concentration. The mainconsideration, of course, when choosing the optimal annealingtemperature is the melting temperature (Tm) of the primers and probes(if used). The annealing temperature chosen for a PCR depends directlyon length and composition of the primers. This is the result of thedifference of hydrogen bonds between A-T (2 bonds) and G-C (3 bonds). Anannealing temperature about 5 degrees below the lowest Tm of the pair ofprimers is usually used.

The final step of PCR amplification is the DNA extension from theprimers which is done by the thermostable Taq DNA polymerase usually at72° C., which is the optimal temperature for the polymerase to work. Thelength of the incubation at each temperature, the temperaturealterations and the number of cycles are controlled by a programmablethermal cycler. The analysis of the PCR products depends on the type ofPCR applied. If a conventional PCR is used, the PCR product is detectedusing for example agarose gel electrophoresis or other polymer gel likepolyacrylamide gels and ethidium bromide (or other nucleic acidstaining).

Conventional RT-PCR is a time-consuming technique with importantlimitations when compared to real time PCR techniques. This combinedwith the fact that ethidium bromide has low sensitivity, yields resultsthat are not always reliable. Moreover, there is an increasedcross-contamination risk of the samples since detection of the PCRproduct requires the post-amplification processing of the samples.Furthermore, the specificity of the assay is mainly determined by theprimers, which can give false-positive results. However, the mostimportant issue concerning conventional RT-PCR is the fact that it is asemi or even a low quantitative technique, where the amplicon can bevisualized only after the amplification ends.

Real time RT-PCR provides a method where the amplicons can be visualizedas the amplification progresses using a fluorescent reporter molecule.There are three major kinds of fluorescent reporters used in real timeRT-PCR, general nonspecific DNA Binding Dyes such as SYBR Green I,TaqMan Probes and Molecular Beacons (including Scorpions).

The real time PCR thermal cycler has a fluorescence detection threshold,below which it cannot discriminate the difference between amplificationgenerated signal and background noise. On the other hand, thefluorescence increases as the amplification progresses and theinstrument performs data acquisition during the annealing step of eachcycle. The number of amplicons will reach the detection baseline after aspecific cycle, which depends on the initial concentration of the targetDNA sequence. The cycle at which the instrument can discriminate theamplification generated fluorescence from the background noise is calledthe threshold cycle (Ct). The higher is the initial DNA concentration,the lower its Ct will be.

Other forms of nucleic acid detection can include next generationsequencing methods such as DNA SEQ or RNA SEQ using any known sequencingplatform including, but not limited to: Roche 454, Solexa GenomeAnalyzer, AB SOLiD, Illumina GA/HiSeq, Ion PGM, Mi Seq, among others(Liu et al, 2012 Journal of Biomedicine and Biotechnology Volume 2012 ID251364; Franca et al., 2002 Quarterly Reviews of Biophysics 35 pg.169-200; Mardis 2008 Genomics and Human Genetics vol. 9 pg. 387-402).

In other embodiments, nucleic acids may be detected with other highthroughput hybridization technologies including microarrays, gene chips,LNA probes, nanoStrings, and fluorescence polarization detection amongothers.

In some embodiments, detection of markers can be achieved at an earlystage of plant growth by harvesting a small tissue sample (e.g., branch,or leaf disk). This approach is preferable when working with largepopulations as it allows breeders to weed out undesirable progeny at anearly stage and conserve growth space and resources for progeny whichshow more promise. In some embodiments the detection of markers isautomated, such that the detection and storage of marker data is handledby a machine. Recent advances in robotics have also led to full serviceanalysis tools capable of handling nucleic acid/protein markerextractions, detection, storage and analysis.

Quantitative Trait Loci

Breeding schemes of the present application can include crosses betweendonor and recipient plants. In some embodiments, said donor plantscontain a gene or genes of interest which may confer the plant with adesirable phenotype. The recipient line can be an elite line havingcertain favorable traits for commercial production. In one embodiment,the elite line may contain other genes that also impart said line withthe desired phenotype. When crossed together, the donor and recipientplant may create a progeny plant with combined desirable loci which mayprovide quantitatively additive effect of a particular characteristic.In that case, QTL mapping can be involved to facilitate the breedingprocess.

A QTL (quantitative trait locus) mapping can be applied to determine theparts of the donor plant's genome conferring the desirable phenotype,and facilitate the breeding methods. Inheritance of quantitative traitsor polygenic inheritance refers to the inheritance of a phenotypiccharacteristic that varies in degree and can be attributed to theinteractions between two or more genes and their environment. Though notnecessarily genes themselves, quantitative trait loci (QTLs) arestretches of DNA that are closely linked to the genes that underlie thetrait in question. QTLs can be molecularly identified to help mapregions of the genome that contain genes involved in specifying aquantitative trait. This can be an early step in identifying andsequencing these genes.

Typically, QTLs underlie continuous traits (those traits that varycontinuously, e.g. yield, height, level of resistance to virus, etc.) asopposed to discrete traits (traits that have two or several charactervalues, e.g. smooth vs. wrinkled peas used by Mendel in hisexperiments). Moreover, a single phenotypic trait is usually determinedby many genes. Consequently, many QTLs are associated with a singletrait.

A quantitative trait locus (QTL) is a region of DNA that is associatedwith a particular phenotypic trait. Knowing the number of QTLs thatexplains variation in the phenotypic trait tells about the geneticarchitecture of a trait. It may tell that a trait is controlled by manygenes of small effect, or by a few genes of large effect or by a severalgenes of small effect and few genes of larger effect.

Another use of QTLs is to identify candidate genes underlying a trait.Once a region of DNA is identified as contributing to a phenotype, itcan be sequenced. The DNA sequence of any genes in this region can thenbe compared to a database of DNA for genes whose function is alreadyknown.

In a recent development, classical QTL analyses are combined with geneexpression profiling i.e. by DNA microarrays. Such expression QTLs(e-QTLs) describes cis- and trans-controlling elements for theexpression of often disease-associated genes. Observed epistatic effectshave been found beneficial to identify the gene responsible by across-validation of genes within the interacting loci with metabolicpathway and scientific literature databases.

QTL mapping is the statistical study of the alleles that occur in alocus and the phenotypes (physical forms or traits) that they produce(see, Meksem and Kahl, The handbook of plant genome mapping: genetic andphysical mapping, 2005, Wiley-VCH, ISBN 3527311165, 9783527311163).Because most traits of interest are governed by more than one gene,defining and studying the entire locus of genes related to a trait giveshope of understanding what effect the genotype of an individual mighthave in the real world.

Statistical analysis is required to demonstrate that different genesinteract with one another and to determine whether they produce asignificant effect on the phenotype. QTLs identify a particular regionof the genome as containing one or several genes, i.e. a cluster ofgenes that is associated with the trait being assayed or measured. Theyare shown as intervals across a chromosome, where the probability ofassociation is plotted for each marker used in the mapping experiment.

To begin, a set of genetic markers must be developed for the species inquestion. A marker is an identifiable region of variable DNA. Biologistsare interested in understanding the genetic basis of phenotypes(physical traits). The aim is to find a marker that is significantlymore likely to co-occur with the trait than expected by chance, that is,a marker that has a statistical association with the trait. Ideally,they would be able to find the specific gene or genes in question, butthis is a long and difficult undertaking. Instead, they can more readilyfind regions of DNA that are very close to the genes in question. When aQTL is found, it is often not the actual gene underlying the phenotypictrait, but rather a region of DNA that is closely linked with the gene.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes. This can be doneusing BLAST, an online tool that allows users to enter a primarysequence and search for similar sequences within the BLAST database ofgenes from various organisms.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is shaped by many independent loci, or by a few loci, andhow those loci interact. This can provide information on how thephenotype may be evolving.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, SNPs, microsatellites, AFLP). Alldifferences between two parental genotypes will segregate in a mappingpopulation based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes are generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency usually corresponds to a low distance betweenmarkers on a chromosome. Comparing all recombination frequencies willresult in the most logical order of the molecular markers on thechromosomes. This most logical order can be depicted in a linkage map(Paterson, 1996, Genome Mapping in Plants. R. G. Landes, Austin.). Agroup of adjacent or contiguous markers on the linkage map that isassociated to a reduced disease incidence and/or a reduced lesion growthrate pinpoints the position of a QTL.

The nucleic acid sequence of a QTL may be determined by methods known tothe skilled person. For instance, a nucleic acid sequence comprisingsaid QTL or a resistance-conferring part thereof may be isolated from adonor plant by fragmenting the genome of said plant and selecting thosefragments harboring one or more markers indicative of said QTL.Subsequently, or alternatively, the marker sequences (or parts thereof)indicative of said QTL may be used as (PCR) amplification primers, inorder to amplify a nucleic acid sequence comprising said QTL from agenomic nucleic acid sample or a genome fragment obtained from saidplant. The amplified sequence may then be purified in order to obtainthe isolated QTL. The nucleotide sequence of the QTL, and/or of anyadditional markers comprised therein, may then be obtained by standardsequencing methods.

One or more such QTLs associated with a desirable trait in a donor plantcan be transferred to a recipient plant to incorporate the desirabletrait into progeny plants by transferring and/or breeding methods.

In one embodiment, an advanced backcross QTL analysis (AB-QTL) is usedto discover the nucleotide sequence or the QTLs responsible for theresistance of a plant. Such method was proposed by Tanksley and Nelsonin 1996 (Tanksley and Nelson, 1996, Advanced backcross QTL analysis: amethod for simultaneous discovery and transfer of valuable QTL fromun-adapted germplasm into elite breeding lines. Theor Appl Genet92:191-203) as a new breeding method that integrates the process of QTLdiscovery with variety development, by simultaneously identifying andtransferring useful QTL alleles from un-adapted (e.g., land races, wildspecies) to elite germplasm, thus broadening the genetic diversityavailable for breeding. AB-QTL strategy was initially developed andtested in tomato, and has been adapted for use in other crops includingrice, maize, wheat, pepper, barley, and bean. Once favorable QTL allelesare detected, only a few additional marker-assisted generations arerequired to generate near isogenic lines (NILs) or introgression lines(ILs) that can be field tested in order to confirm the QTL effect andsubsequently used for variety development.

Isogenic lines in which favorable QTL alleles have been fixed can begenerated by systematic backcrossing and introgressing of marker-defineddonor segments in the recurrent parent background. These isogenic linesare referred to as near isogenic lines (NILs), introgression lines(ILs), backcross inbred lines (BILs), backcross recombinant inbred lines(BCRIL), recombinant chromosome substitution lines (RCSLs), chromosomesegment substitution lines (CSSLs), and stepped aligned inbredrecombinant strains (STAIRSs). An introgression line in plant molecularbiology is a line of a crop species that contains genetic materialderived from a similar species. ILs represent NILs with relatively largeaverage introgression length, while BILs and BCRILs are backcrosspopulations generally containing multiple donor introgressions per line.As used herein, the term “introgression lines or ILs” refers to plantlines containing a single marker defined homozygous donor segment, andthe term “pre-ILs” refers to lines which still contain multiplehomozygous and/or heterozygous donor segments.

To enhance the rate of progress of introgression breeding, a geneticinfrastructure of exotic libraries can be developed. Such an exoticlibrary comprises a set of introgression lines, each of which has asingle, possibly homozygous, marker-defined chromosomal segment thatoriginates from a donor exotic parent, in an otherwise homogenous elitegenetic background, so that the entire donor genome would be representedin a set of introgression lines. A collection of such introgressionlines is referred as libraries of introgression lines or IL libraries(ILLs). The lines of an ILL cover usually the complete genome of thedonor, or the part of interest. Introgression lines allow the study ofquantitative trait loci, but also the creation of new varieties byintroducing exotic traits. High resolution mapping of QTL using ILLsenable breeders to assess whether the effect on the phenotype is due toa single QTL or to several tightly linked QTL affecting the same trait.In addition, sub-ILs can be developed to discover molecular markerswhich are more tightly linked to the QTL of interest, which can be usedfor marker-assisted breeding (MAB). Multiple introgression lines can bedeveloped when the introgression of a single QTL is not sufficient toresult in a substantial improvement in agriculturally important traits(Gur and Zamir, Unused natural variation can lift yield barriers inplant breeding, 2004, PLoS Biol.; 2(10):e245).

Plant Transformation

In some embodiments, the present invention provides transformed sweetcorn plants or parts thereof that have been transformed so that itsgenetic material contains one or more transgenes, preferably operablylinked to one or more regulatory elements. Also, the invention providesmethods for producing a sweet corn plant containing in its geneticmaterial one or more transgenes, preferably operably linked to one ormore regulatory elements, by crossing transformed sweet corn plants witha second plant of another sweet corn, so that the genetic material ofthe progeny that results from the cross contains the transgene(s),preferably operably linked to one or more regulatory elements. Theinvention also provides methods for producing a sweet corn plant thatcontains in its genetic material one or more transgene(s), wherein themethod comprises crossing a sweet corn with a second plant of anothersweet corn which contains one or more transgene(s) operably linked toone or more regulatory element(s) so that the genetic material of theprogeny that results from the cross contains the transgene(s) operablylinked to one or more regulatory element(s). Transgenic sweet cornplants, or parts thereof produced by the method are in the scope of thepresent invention.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed sweet corn hybrid plant.

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

i. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985), Jefferson et al., Embo J.3901-390764, (1987), Diant, et al., Molecular Breeding, 3:1, 75-86(1997), Valles et al., PI. Cell. Rep. 145-148:13 (1984). A. tumefaciensand A. rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems andmethods for Agrobacterium-mediated gene transfer are provided by Gruberet al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7,1997.

ii. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has been achieved in rice and corn. Hiei et al., ThePlant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616 issued Jan.7, 1997. Several methods of plant transformation, collectively referredto as direct gene transfer, have been developed as an alternative toAgrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 micron. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Russell, D. R.,et al., Pl. Cell. Rep., 12, 165-169 (1993); Aragao, F. J. L., et al.,Plant Mol. Biol., 20, 357-359 (1992); Aragao, Theor. Appl. Genet.,93:142-150 (1996); Kim, J.; Minamikawa, T., Plant Science, 117:131-138(1996); Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J. C.,Trends Biotech. 6:299 (1988), Klein et al., BioTechnology 6:559-563(1988), Sanford, J. C., Physiol Plant 7:206 (1990), Klein et al.,BioTechnology 10:268 (1992). Gray et al., Plant Cell Tissue and OrganCulture. 1994, 37:2, 179-184.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., BioTechnology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. D'Halluin et al., Plant Cell 4:1495-1505 (1992) andSpencer et al., Plant Mol. Biol. 24:51-61 (1994).

Any DNA sequence(s), whether from a different species or from the samespecies that is inserted into the genome using transformation isreferred to herein collectively as “transgenes”. In some embodiments ofthe invention, a transformed variant of sweet corn hybrid may contain atleast one transgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 transgenes. In another embodiment of the invention, atransformed variant of the another sweet corn plant used as the donorline may contain at least one transgene but could contain at least 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 transgenes.

Following transformation of sweet corn target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line or a transgenic hybrid plant. Thetransgenic inbred line could then be crossed, with another(non-transformed or transformed) inbred line, in order to produce a newtransgenic inbred line or plant. Alternatively, a genetic trait whichhas been engineered into a particular sweet corn plant using theforegoing transformation techniques could be moved into another sweetcorn plant using traditional backcrossing techniques that are well knownin the plant breeding arts. For example, a backcrossing approach couldbe used to move an engineered trait from a public, non-elite inbred lineinto an elite inbred line, or from an inbred line containing a foreigngene in its genome into an inbred line or lines which do not containthat gene. As used herein, “crossing” can refer to a simple X by Ycross, or the process of backcrossing, depending on the context.

iii. Selection

For efficient plant transformation, a selection method must be employedsuch that whole plants are regenerated from a single transformed celland every cell of the transformed plant carries the DNA of interest.These methods can employ positive selection, whereby a foreign gene issupplied to a plant cell that allows it to utilize a substrate presentin the medium that it otherwise could not use, such as mannose or xylose(for example, refer U.S. Pat. Nos. 5,767,378; 5,994,629). Moretypically, however, negative selection is used because it is moreefficient, utilizing selective agents such as herbicides or antibioticsthat either kill or inhibit the growth of non-transformed plant cellsand reducing the possibility of chimeras. Resistance genes that areeffective against negative selective agents are provided on theintroduced foreign DNA used for the plant transformation. For example,one of the most popular selective agents used is the antibiotickanamycin, together with the resistance gene neomycin phosphotransferase(nptII), which confers resistance to kanamycin and related antibiotics(see, for example, Messing & Vierra, Gene 19: 259-268 (1982); Bevan etal., Nature 304:184-187 (1983)). However, many different antibiotics andantibiotic resistance genes can be used for transformation purposes(refer U.S. Pat. Nos. 5,034,322, 6,174,724 and 6,255,560). In addition,several herbicides and herbicide resistance genes have been used fortransformation purposes, including the bar gene, which confersresistance to the herbicide phosphinothricin (White et al., Nucl AcidsRes 18: 1062 (1990), Spencer et al., Theor Appl Genet 79: 625-631(1990),U.S. Pat. Nos. 4,795,855, 5,378,824 and 6,107,549). In addition, thedhfr gene, which confers resistance to the anticancer agentmethotrexate, has been used for selection (Bourouis et al., EMBO J.2(7): 1099-1104 (1983).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include beta-glucuronidase (GUS,beta-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984), Valles et al., Plant CellReport 3:3-4 145-148 (1994), Shetty et al., Food Biotechnology 11:2111-128 (1997)

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells. Chalfieet al., Science 263:802 (1994). GFP and mutants of GFP may be used asscreenable markers.

iv. Expression Vectors

Genes can be introduced in a site directed fashion using homologousrecombination. Homologous recombination permits site-specificmodifications in endogenous genes and thus inherited or acquiredmutations may be corrected, and/or novel alterations may be engineeredinto the genome. Homologous recombination and site-directed integrationin plants are discussed in, for example, U.S. Pat. Nos. 5,451,513;5,501,967 and 5,527,695.

The expression control elements used to regulate the expression of agiven protein can either be the expression control element that isnormally found associated with the coding sequence (homologousexpression element) or can be a heterologous expression control element.A variety of homologous and heterologous expression control elements areknown in the art and can readily be used to make expression units foruse in the present invention. Transcription initiation regions, forexample, can include any of the various opine initiation regions, suchas octopine, mannopine, nopaline and the like that are found in the Tiplasmids of Agrobacterium tumefaciens. Alternatively, plant viralpromoters can also be used, such as the cauliflower mosaic virus 19S and35S promoters (CaMV 19S and CaMV 35S promoters, respectively) to controlgene expression in a plant (U.S. Pat. Nos. 5,352,605; 5,530,196 and5,858,742 for example). Enhancer sequences derived from the CaMV canalso be utilized (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938;5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example). Lastly,plant promoters such as prolifera promoter, fruit specific promoters,Ap3 promoter, heat shock promoters, seed specific promoters, etc. canalso be used.

Either a gamete-specific promoter, a constitutive promoter (such as theCaMV or Nos promoter), an organ-specific promoter (such as the E8promoter from tomato), or an inducible promoter is typically ligated tothe protein or anti sense encoding region using standard techniquesknown in the art. The expression unit may be further optimized byemploying supplemental elements such as transcription terminators and/orenhancer elements.

Thus, for expression in plants, the expression units will typicallycontain, in addition to the protein sequence, a plant promoter region, atranscription initiation site and a transcription termination sequence.Unique restriction enzyme sites at the 5′ and 3′ ends of the expressionunit are typically included to allow for easy insertion into apre-existing vector.

In the construction of heterologous promoter/structural gene or antisense combinations, the promoter is preferably positioned about the samedistance from the heterologous transcription start site as it is fromthe transcription start site in its natural setting. As is known in theart, however, some variation in this distance can be accommodatedwithout loss of promoter function.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes. If the mRNA encoded by the structural gene is tobe efficiently processed, DNA sequences which direct polyadenylation ofthe RNA are also commonly added to the vector construct. Polyadenylationsequences include, but are not limited to the Agrobacterium octopinesynthase signal (Gielen et al., EMBO J 3:835-846 (1984)) or the nopalinesynthase signal (Depicker et al., Mol. and Appl. Genet. 1:561-573(1982)). The resulting expression unit is ligated into or otherwiseconstructed to be included in a vector that is appropriate for higherplant transformation. One or more expression units may be included inthe same vector. The vector will typically contain a selectable markergene expression unit by which transformed plant cells can be identifiedin culture. Usually, the marker gene will encode resistance to anantibiotic, such as G418, hygromycin, bleomycin, kanamycin, orgentamicin or to an herbicide, such as glyphosate (Round-Up) orglufosinate (BASTA) or atrazine. Replication sequences, of bacterial orviral origin, are generally also included to allow the vector to becloned in a bacterial or phage host; preferably a broad host range forprokaryotic origin of replication is included. A selectable marker forbacteria may also be included to allow selection of bacterial cellsbearing the desired construct. Suitable prokaryotic selectable markersinclude resistance to antibiotics such as ampicillin, kanamycin ortetracycline. Other DNA sequences encoding additional functions may alsobe present in the vector, as is known in the art. For instance, in thecase of Agrobacterium transformations, T-DNA sequences will also beincluded for subsequent transfer to plant chromosomes.

To introduce a desired gene or set of genes by conventional methodsrequires a sexual cross between two lines, and then repeatedback-crossing between hybrid offspring and one of the parents until aplant with the desired characteristics is obtained. This process,however, is restricted to plants that can sexually hybridize, and genesin addition to the desired gene will be transferred.

Recombinant DNA techniques allow plant researchers to circumvent theselimitations by enabling plant geneticists to identify and clone specificgenes for desirable traits, such as improved fatty acid composition, andto introduce these genes into already useful varieties of plants. Oncethe foreign genes have been introduced into a plant, that plant can thenbe used in conventional plant breeding schemes (e.g., pedigree breeding,single-seed-descent breeding schemes, reciprocal recurrent selection) toproduce progeny which also contain the gene of interest.

v. Promoters

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain organs,such as leaves, roots, seeds and tissues such as fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred”. Promoters which initiate transcription only incertain tissue are referred to as “tissue-specific”. A “cell type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

A) Inducible Promoters

An inducible promoter is operably linked to a gene for expression insweet corn. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in sweet corn. With an inducible promoter therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners (Gatzet al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10(Gatz et al., Mol. Gen. Genetics 227:229-237 (1991)). A particularlypreferred inducible promoter is a promoter that responds to an inducingagent to which plants do not normally respond. An exemplary induciblepromoter is the inducible promoter from a steroid hormone gene, thetranscriptional activity of which is induced by a glucocorticosteroidhormone (Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991)).

B) Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression insweet corn or the constitutive promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in sweet corn.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, Xba1/Nco1 fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXba1/Nco1 fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.C.

C) Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin sweet corn. Optionally, the tissue-specific promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in sweet corn. Plantstransformed with a gene of interest operably linked to a tissue-specificpromoter produce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al.,Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129(1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol.108:1657 (1989), Creissen et al., Plant 1 2:129 (1991), Kalderon, etal., Cell 39:499-509 (1984), Stiefel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a one embodiment, the transgenic plant provided forcommercial production of foreign protein is a sweet corn plant. Inanother preferred embodiment, the biomass of interest is seed or cob.For the relatively small number of transgenic plants that show higherlevels of expression, a genetic map can be generated, primarily viaconventional RFLP, PCR and SSR analysis, which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Glick and Thompson, Methodsin Plant Molecular Biology and Biotechnology, Glick and Thompson Eds.,CRC Press, Boca Raton 269:284 (1993). Map information concerningchromosomal location is useful for proprietary protection of a subjecttransgenic plant. If unauthorized propagation is undertaken and crossesmade with other germplasm, the map of the integration region can becompared to similar maps for suspect plants, to determine if the latterhave a common parentage with the subject plant. Map comparisons wouldinvolve hybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

Examples of Genes that Confer Resistance to Pests or Disease and thatEncode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with one ormore cloned resistance gene to engineer plants that are resistant tospecific pathogen strains. See, for example Jones et al., Science266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Ptogene for resistance to Pseudomonas syringae pv. tomato encodes a proteinkinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2 genefor resistance to Pseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btalpha-endotoxin gene. Moreover, DNA molecules encoding alpha-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus alpha-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. Pratt et al., Biochem.Biophys. Res. Comm. 163:1243 (1989) (an allostatin is identified inDiploptera puntata). See also U.S. Pat. No. 5,266,317 to Tomalski etal., who disclose genes encoding insect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci. 89:43 (1993),of heterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-alpha-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-alpha-1, 4-D-galacturonase. See Lamb et al.,BioTechnology 10:1436 (1992). The cloning and characterization of a genewhich encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

R. A developmental-arrestee protein produced in nature by a plant. Forexample, Logemann et al., BioTechnology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant 5-enolpyruvlshikimate-3phosphate synthase (EPSP) and aroA genes, respectively) and otherphosphono compounds such as glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus PAT, bar, genes), andpyridinoxy or phenoxy propionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession number 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclosenucleotide sequences of glutamine synthatase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a PAT gene is provided in European application No. 0 242 246to Leemans et al. DeGreef et al., BioTechnology 7:61 (1989), describethe production of transgenic plants that express chimeric bar genescoding for PAT activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knutzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992)

B. Increased resistance to high light stress such as photo-oxidativedamages, for example by transforming a plant with a gene coding for aprotein of the Early Light Induced Protein family (ELIP) as described inWO 03/074713 in the name of Biogemma.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bact. 170:810 (1988)(nucleotide sequence of Streptococcus mutants fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 20:220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,BioTechnology 10:292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis .alpha.-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al., PlantPhysiol. 102:1045 (1993) (maize endosperm starch branching enzyme II).

D. Increased resistance/tolerance to water stress or drought, forexample, by transforming a plant to create a plant having a modifiedcontent in ABA-Water-Stress-Ripening-Induced proteins (ARS proteins) asdescribed in WO 01/83753 in the name of Biogemma, or by transforming aplant with a nucleotide sequence coding for a phosphoenolpyruvatecarboxylase as shown in WO 02/081714. The tolerance of corn to droughtcan also be increased by an overexpression of phosphoenolpyruvatecarboxylase (PEPC-C4), obtained, for example from sorghum.

E. Increased content of cysteine and glutathione, useful in theregulation of sulfur compounds and plant resistance against variousstresses such as drought, heat or cold, by transforming a plant with agene coding for an Adenosine 5′ Phosphosulfate as shown in WO 01/49855.

F. Increased nutritional quality, for example, by introducing a zeingene which genetic sequence has been modified so that its proteinsequence has an increase in lysine and proline. The increasednutritional quality can also be attained by introducing into the maizeplant an albumin 2S gene from sunflower that has been modified by theaddition of the KDEL peptide sequence to keep and accumulate the albuminprotein in the endoplasmic reticulum.

G. Decreased phytate content: 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

Tissue Culture

As it is well known in the art, tissue culture of sweet corn can be usedfor the in vitro regeneration of sweet corn plants. Tissues cultures ofvarious tissues of sweet corn and regeneration of plants therefrom arewell known and published. By way of example, a tissue culture comprisingorgans has been used to produce regenerated plants as described inGirish-Chandel et al., Advances in Plant Sciences. 2000, 13: 1, 11-17,Costa et al., Plant Cell Report. 2000, 19: 3327-332, Plastira et al.,Acta Horticulturae. 1997, 447, 231-234, Zagorska et al., Plant CellReport. 1998, 17: 12 968-973, Asahura et al., Breeding Science. 1995,45: 455-459, Chen et al., Breeding Science. 1994, 44: 3, 257-262, Patilet al., Plant and Tissue and Organ Culture. 1994, 36: 2, 255-258. It isclear from the literature that the state of the art is such that thesemethods of obtaining plants are routinely used and have a very high rateof success. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce sweet corn plants havingall the physiological and morphological characteristics of hybrid sweetcorn plant AZLAN.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollens, flowers, seeds, leaves,stems, roots, root tips, anthers, pistils, meristematic cells, axillarybuds, ovaries, seed coats, endosperms, hypocotyls, cotyledons and thelike. Means for preparing and maintaining plant tissue culture are wellknown in the art. By way of example, a tissue culture comprising organshas been used to produce regenerated plants. U.S. Pat. Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

EXAMPLES

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Example 1—Development of New AZLAN Sweet Corn Variety

Breeding History of AZLAN

Hybrid sweet corn plant AZLAN has superior characteristics. The female(SCF7) and male (SCM8) parents were crossed to produce hybrid (F1) seedsof AZLAN. The seeds of AZLAN can be grown to produce hybrid plants andparts therefor. The hybrid AZLAN can be propagated by seeds by crossingsweet corn inbred line SCF7 with sweet corn inbred line SCM8 orvegetatively.

The origin and breeding history of hybrid plant AZLAN can be summarizedas follows: the line SCF7 (a proprietary line by HM.CLAUSE, Inc.) wasused as the female plant and crossed using pollen from SCM8 (aproprietary line owned by HM.CLAUSE, Inc.). The first trial planting ofthis hybrid was done in Stevens Point, Wis., in the summer of the firstyear of development. The hybrid was further trialed for two additionalyears in California, Arizona.

The inbred line SCF7 is a white shrunken-2 inbred line was used as thefemale parent in this cross. The inbred line SCF7 has good performancein seed production, a well-proportioned ear, resistance to Pucciniasorghi (Rp1g) and a high level of eating quality.

The inbred line SCM8 is a white shrunken-2 inbred line used as the maleparent in this cross. The inbred line SCM8 has a strong plant type, darkgreen plant, husk leaves and flag leaves; and has a high level of eatingquality.

Hybrid sweet corn plant AZLAN is similar to hybrid sweet corn plantTurbo. Turbo is a commercial variety. As shown in Tables 1, whilesimilar to hybrid sweet corn plant Turbo, there are significantdifferences including the plant height which is 86.3 inches for AZLANwhile the plant height for Turbo is 84.4 inches, the ear height frombase of top ear node which is 31.8 inches for AZLAN while Turbo is 29.8inches. The cob diameter for AZLAN is 4.8 cm while Turbo is 4.6 cm.Also, AZLAN has a brix score of 22.5 while the brix score of Turbo is21.8

Some of the criteria used to select the hybrid AZLAN as well as theinbred parent lines in various generations include: strong plant, highyield, husk extension, ear length, ear diameter and a high level ofeating quality with a high level of sweetness.

Hybrid sweet corn plant AZLAN has shown uniformity and stability for thetraits, within the limits of environmental influence for the traits asdescribed in the following Variety Descriptive Information. No varianttraits have been observed or are expected for important agronomicaltraits in sweet corn hybrid AZLAN.

Hybrid sweet corn plant AZLAN has the following morphologic and othercharacteristics, as compared to Turbo (based primarily on data collectedin Wisconsin, all experiments done under the direct supervision of theapplicant).

TABLE 1 WHEN TRAIT DESCRIPTION AZLAN Turbo Type Sweet, sugary-1 (su1)Sweet, sugary-1 (su1) Color Yellow Yellow Region where developed in theUSA Midwest Midwest Flowering from planting to 50% of plants in silk 67d 68 d Flowering from planting to 50% of plants in pollen 66 d 67 dPlant Traits Fresh plant height (to tassel tip); average 10 samples 86.3″  84.4″ harvest Fresh ear height (to base of top ear node);average 10  31.8″  29.8″ harvest samples Fresh length of top earinternode; average 10 samples  6.5″  6.6″ harvest Fresh anthocyanin ofbrace roots; absent, weak, Medium Weak harvest medium, strong, very'strong Leaf Traits Emergence first leaf: anthocyanin coloration ofsheath; Medium Medium absent, weak, medium, strong, very strongEmergence first leaf: shape of tip; pointed, pointed to Rounded toRounded to rounded, rounded to spatulate, spatulate spatulate spatulateFlowering foliage intensity of green color; light, medium, Medium Mediumdark Flowering number of leaves above top ear; average 10  5.7  5.4samples Fresh width of ear node leaf; average 10 samples  3.2″  3.5″harvest Fresh length of ear node leaf in inches; average 10  32.7″ 33″harvest samples Flowering angle between blade and stem (on leaf justabove upper ear) 1-9 1 very small, 3 small +/= 25*, 5 med +/= 50*, 5 5 7large +/= 75*, 9 very large >= 90* Flowering leaf undulation of marginof blade; absent, Intermediate Intermediate intermediate, strongFlowering degrees leaf angle (curvature) absent slight recurved modrecurved strongly Moderately Moderately recurved v strongly recurvedrecurved recurved Flowering sheath pubescence (1 = none to 9 = likepeach 9 8 fuzz) Flowering leaf: anthocyanin coloration of sheath (inmiddle of plant) absent weak medium strong very strong Absent AbsentFlowering anthocyanm coloration of internodes (in middle of plant)absent weak medium strong very strong Absent Absent Flowering stem:degree of zig-zag; absent, slight, strong Absent Slight Tassel TraitsFlowering branch angle from central spike 1 very small, 3 small +/= 25*,5 med +/= 50*, 7 9 7 large +/= 75*, 9 very large >= 90* Flowering anglebetween main axis and lateral branches (in lower third of tassel) 1 verysmall. 3 small +/= 25*, 5 med +/= 50*, 7 9 7 large +/= 75*, 9 verylarge >= 90* Flowering attitude of lateral branches (in lower third oftassel) absent slight recurved mod recurved strongly Absent Absentrecurved v strongly recurved Flowering length of lateral branches; veryshort, short,  7.9″  8.6″ medium, long, very long Fresh length (from topleaf collar to tassel tip);  13.7″  14.3″ harvest average 10 samplesFlowering length above lowest lateral branch; average 10  12.9″  13.9″samples Flowering length above highest lateral branch; average 10 10″ 9.1″ samples Flowering anther color; yellow, green, red, pink Pink PinkFlowering glume color; Red Red Flowering bar glumes (glume bands);absent, present Absent Absent Flowering anthocyanin coloration at baseof glumes; Absent Absent absent, weak, medium, strong, very strongFlowering anthocyanin coloration of glumes excluding Medium Weak base;absent, weak, medium, strong, very strong Flowering anthocyanincoloration of anthers (middle third, fresh anthers) absent weak mediumstrong very strong Weak Medium Flowering ear: anthocyanin coloration ofsilk; absent, Absent Absent present Flowering ear: intensity ofanthocyanin coloration of Absent Absent silks; absent, weak, medium,strong, very strong Ear Traits Flowering (unhusked data) silk color (3days after Yellow Yellow emergence); yellow, green, pink, red Fresh(unhusked data) husk tightness (1 = very loose 5 4 harvest to 9 = verytight) Fresh (unhusked data) husk extension (at harvest) in  1.6″  1.4″harvest inches Fresh (husked ear data) ear length; average 10 21 cm 20.7cm harvest samples Fresh (husked ear data) ear diameter at mid-point,4.8 cm 4.6 cm harvest average 10 samples Fresh (husked ear data) numberof kernel rows, 18  18  harvest average Fresh (husked ear data) rowalignment (1 to 9) 6 7 harvest Fresh (husked ear data) shank length ininches  2.6″  2.7″ harvest Fresh (husked ear data) ear taper; conical,conical Conical Conical harvest cylindrical, cylindrical cylindricalcylindrical Fresh number of kernel colors; one or two One One harvestFresh intensity of yellow color; light, medium, dark Medium Lightharvest Fresh fresh kernel width mm 8.7 mm 7.5 mm harvest Fresh freshkernel depth (mm) 11.4 mm 11.4 mm harvest Pre endosperm type; sh2 or su1su1 su1 Planting Fresh brix; average 10 samples 22.5 21.8 harvest CobTraits Fresh diameter at mid-point; average 10 samples 2.8 cm 2.2 cmharvest Fresh cob color White White harvest Disease Reaction TraitsFresh Common Rust (Puccinia sorghi) Resistant Resistant harvest FreshMaize Dwarf Mosaic Virus (MDMV) Resistant Resistant harvest FreshNorthern Corn Leaf Blight (Exserohilum Intermediate Intermediate harvestturcicum) Resistant Resistant

In Tables 2 and 3, the traits and characteristics of hybrid sweet cornplant AZLAN are compared to the variety Turbo. The data was collectedduring two growing season from several field locations in the UnitedStates, all experiments done under the direct supervision of theapplicant.

In Table 2, the first column shows the “Variety Name”. The second columnshows the “Trial Location” of the testing. The third column shows the“Ear Length” in centimeters (cm). The fourth column shows the “EarDiameter” in centimeters (cm). The fifth column is the “Tip Fill” and isgiven in centimeters (cm) without fill. The sixth column “Recovery” isthe weight of corn kernels cut from the cob compared to total harvestweight of the ears and expressed as a %. The seventh column give the“Average Ear Weight” including husk in pounds (lbs).

TABLE 2 Ear Ear Average Ear Variety TRIAL Length Diameter Tip FillRecovery Weight Name LOCATION (cm) (cm) (cm < 0) (%) (lbs) AZLAN SunPrairie, 23.5  5.2 cm −0.5 cm 52% 0.93 lbs Wisconsin Turbo Sun Prairie,22 cm 5 cm −0.5 cm 53% 0.89 lbs Wisconsin

In Table 3, the first column shows the “Variety Name”. The second columnshows the “Trial Location” of the testing. The third column shows the“Ear Length” in centimeters (cm). The fourth column shows the “EarDiameter” in centimeters (cm). The fifth column is the “Tip Fill” and isgiven in centimeters (cm) without fill. The sixth column “Number ofRows” is the total number of kernels rows around the diameter of thecob. The seventh column is the “Kernel Depth” in centimeters (cm), anaverage of the length or depth of the kernel from outer face to thecutting point near the cob. In the eight column the average length ofthe husk above the tip of the ear is given in centimeters (cm).

TABLE 3 Ear Ear Kernel Husk Variety Trial Length Diameter Tip FillNumber of Depth Cover Name Location (cm) (cm) (cm < 0) Rows (cm) (cm)AZLAN Tartas, 20 cm 5.7 cm 0 18 1.3 cm 2 cm France Turbo Tartas, 21 cm5.5 cm 0 16 1.5 cm 2 cm France

DEPOSIT INFORMATION

A deposit of the sweet corn seed of this invention is maintained byHM.CLAUSE, Inc. HM.CLAUSE, Sun Prairie Research & Development, 1677Muller Road, Sun Prairie, Wis. 53590 USA. In addition, a sample of thehybrid sweet corn seed of this invention has been deposited with theNational Collections of Industrial, Food and Marine Bacteria (NCIMB),NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen,AB21 9YA Scotland. The deposit for the hybrid sweet corn AZLAN was madeon Nov. 11, 2021.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 CFR 1.801-1.809, Applicants hereby make thefollowing statements regarding the deposited hybrid sweet corn AZLAN(deposited as NCIMB Accession No. 43888).

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;

2. All restrictions on availability to the public will be irrevocablyremoved upon granting of the patent under conditions specified in 37 CFR1.808;

3. The deposit will be maintained in a public repository for a period of30 years or 5 years after the last request or for the effective life ofthe patent, whichever is longer;

4. A test of the viability of the biological material at the time ofdeposit will be conducted by the public depository under 37 CFR 1.807;and

5. The deposit will be replaced if it should ever become unavailable.

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. §122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 625 seeds of thesame variety with the NCIMB.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed is:
 1. A hybrid sweet corn plant designated AZLAN,wherein a representative sample of seed of said hybrid has beendeposited under NCIMB No.
 43888. 2. A part of hybrid sweet corndesignated AZLAN, wherein the part is selected from the group consistingof a leaf, a flower, an ear, a stalk, a root, a rootstock, a scion, apeduncle, a stamen, an anther, a pistil, a pollen, an ovule, and a cell,and wherein a representative sample of seed of said hybrid has beendeposited under NCIMB No.
 43888. 3. A tissue culture of regenerablecells produced from the sweet corn plant or the part thereof of claim 2.4. A sweet corn plant regenerated from the tissue culture of claim 3,said plant having all the physiological and morphologicalcharacteristics of hybrid sweet corn designated AZLAN when grown underthe same environmental conditions and wherein a representative sample ofseed of said hybrid has been deposited under NCIMB No.
 43888. 5. Amethod for harvesting a sweet corn ear, the method comprising: (a)growing the sweet corn plant of claim 1 to produce a sweet corn ear, and(b) harvesting said sweet corn ear.
 6. A method for producing a sweetcorn seed, the method comprising: (a) crossing a first sweet corn plantwith a second sweet corn plant and (b) harvesting the resultant sweetcorn seed, wherein said first sweet corn plant and/or second sweet cornplant is the sweet corn plant of claim
 1. 7. A method for producing asweet corn seed, the method comprising: (a) self-pollinating the sweetcorn plant of claim 1 and (b) harvesting the resultant sweet corn seed.8. A method of vegetatively propagating the sweet corn plant of claim 1,the method comprising: (a) collecting a part capable of being propagatedfrom the plant of claim 1 and (b) regenerating a plant from said part.9. The method of claim 8, further comprising (c) harvesting an ear fromsaid regenerated plant.
 10. A plant obtained from the method of claim 8,wherein said plant has all the physiological and morphologicalcharacteristics of hybrid sweet corn designated AZLAN when grown underthe same environmental conditions.
 11. A method of producing a sweetcorn plant derived from hybrid sweet corn designated AZLAN, the methodcomprising: (a) self-pollinating the plant of claim 1 at least once toproduce a progeny plant.
 12. The method of claim 11, further comprisingthe steps of: (b) crossing the progeny plant derived from the hybridsweet corn designated AZLAN with itself or a second sweet corn plant toproduce a seed of progeny plant of subsequent generation; (c) growingthe progeny plant of the subsequent generation from the seed; (d)crossing the progeny plant of the subsequent generation with itself or asecond sweet corn plant to produce a sweet corn plant derived from thehybrid sweet corn designated AZLAN; and (e) repeating step (b) and/or(c) for at least one generation to produce a sweet corn plant derivedfrom the hybrid sweet corn designated AZLAN.
 13. A method of producing asweet corn plant derived from hybrid sweet corn designated AZLAN, themethod comprising: (a) crossing the plant of claim 1 with a second sweetcorn plant to produce a progeny plant.
 14. The method of claim 13,further comprising the steps of: (b) crossing the progeny plant derivedfrom the hybrid sweet corn plant designated AZLAN with itself or asecond sweet corn plant to produce a seed of progeny plant of subsequentgeneration; (c) growing the progeny plant of the subsequent generationfrom the seed; (d) crossing the progeny plant of the subsequentgeneration with itself or a second sweet corn plant to produce a sweetcorn plant derived from the sweet corn hybrid sweet corn plantdesignated AZLAN; and (e) repeating step (b) and/or (c) to produce asweet corn plant derived from the hybrid sweet corn plant designatedAZLAN.
 15. A method of producing a plant of hybrid sweet corn designatedAZLAN comprising at least one desired trait, the method comprisingintroducing a single locus conversion conferring the desired trait intohybrid sweet corn designated AZLAN, whereby a plant of hybrid sweet corndesignated AZLAN comprising the desired trait is produced.
 16. A sweetcorn plant, comprising a single locus conversion and essentially all thecharacteristics of hybrid sweet corn designated AZLAN deposited underNCIMB No. 43888, wherein the single locus conversion is introduced intothe plant by a genome editing technique with a nuclease selected fromthe group consisting of Zinc finger nuclease (ZFN), TranscriptionActivation-Like Effector Nuclease (TALEN), Clustered RegularlyInterspaced Short Palindromic repeats-associated endonuclease Cas9(CRISPR-Cas9), engineered meganuclease, and engineered homingendonuclease.
 17. The plant of claim 16, wherein the single locusconversion confers said plant with herbicide resistance.
 18. A method ofproducing a sweet corn plant, the method comprising grafting a rootstockor a scion of the hybrid sweet corn plant of claim 1 to another sweetcorn plant.
 19. A method for producing nucleic acids, the methodcomprising isolating nucleic acids from the plant of claim 1, a part, ora cell thereof.
 20. A method for producing a second sweet corn plant,the method comprising applying plant breeding techniques to the plant orpart of claim 1 to produce the second sweet corn plant.